bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-
Toxoplasma
Activities of 24 Quinolones and Fluoroquinolones In Vitro: Prediction of Activity by Molecular Topology and Virtual Computational Techniques
Authors: Gozalbes Rafael , Brun-Pascaud Monique , Garcia-Domenech Ramon , Galvez Jorge , Girard Pierre-Marie , Doucet Jean-Pierre , Derouin Francis
Published: July 2002

The apicoplast, a plastid-like organelle of
Toxoplasma gondii
, is thought to be a unique drug target for quinolones. In this study, we assessed the in vitro activity of quinolones against
T. gondii
and developed new quantitative structure-activity relationship models able to predict this activity. The anti-
Toxoplasma
activities of 24 quinolones were examined by means of linear discriminant analysis (LDA) using topological indices as structural descriptors. In parallel, in vitro 50% inhibitory concentrations (IC
50
s) were determined in tissue culture. A multilinear regression (MLR) analysis was then performed to establish a model capable of classifying quinolones by in vitro activity. LDA and MLR analysis were applied to virtual structures to identify the influence of each atom or substituent of the quinolone ring on anti-
Toxoplasma
activity. LDA predicted that 20 of the 24 quinolones would be active against
T. gondii
. This was confirmed in vitro for most of the quinolones. Trovafloxacin, grepafloxacin, gatifloxacin, and moxifloxacin were the quinolones most potent against
T. gondii
, with IC
50
s of 0.4, 2.4, 4.1, and 5.1 mg/liter, respectively. Using MLR analysis, a good correlation was found between measured and predicted IC
50
s (
r
2
= 0.87, cross-validation
r
2
= 0.74). MLR analysis showed that the carboxylic group at position C-3 of the quinolone ring was not essential for anti-
Toxoplasma
activity. In contrast, activity was totally dependent on the presence of a fluorine at position C-6 and was enhanced by the presence of a methyl group at C-5 or an azabicyclohexane at C-7. A nucleophilic substituent at C-8 was essential for the activity of gatifloxacin and moxifloxacin.
Antibacterial and Antimembrane Activities of Cecropin A in
Escherichia coli
Authors: Silvestro Loraine , Weiser Jeffrey N. , Axelsen Paul H.
Published: July 2002

The ability of cecropin A to permeabilize and depolarize the membranes of
Escherichia coli
ML-35p bacteria has been compared to its bactericidal activity in an extension of earlier studies performed on synthetic lipid vesicle membranes (L. Silvestro, K. Gupta, J. H. Weiser, and P. H. Axelsen, Biochemistry 36:11452–11460, 1997). Our results indicate that differences in the concentration dependences of membrane permeabilization and depolarization seen in synthetic vesicles are not manifested in whole bacteria. The concentration dependences of both phenomena roughly correlate with bactericidal activity, suggesting that the bactericidal mechanism of cecropin A is related to membrane permeabilization.
Towards an Understanding of the Mechanism of Pyrimethamine-Sulfadoxine Resistance in
Plasmodium falciparum
: Genotyping of Dihydrofolate Reductase and Dihydropteroate Synthase of Kenyan Parasites
Authors: Nzila A. M. , Mberu E. K. , Sulo J. , Dayo H. , Winstanley P. A. , Sibley C. H. , Watkins W. M.
Published: July 2002

The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were “pure” triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.
TLA-1: a New Plasmid-Mediated Extended-Spectrum β-Lactamase from
Escherichia coli
Authors: Silva J. , Aguilar C. , Ayala G. , Estrada M. A. , Garza-Ramos U. , Lara-Lemus R. , Ledezma L.
Published: July 2002

Escherichia coli
R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two β-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from
E. coli
R170 to
E. coli
J53-2. The transferred plasmid, RZA92, which encoded a single β-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 β-lactamase (pI 9.0) was cloned from the transconjugant by transformation to
E. coli
DH5α. Sequencing of the
bla
TLA-1
gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A β-lactamases:
70
SXXK,
130
SDN, and
234
KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A β-lactamase from
Chryseobacterium
(
Flavobacterium
)
meningosepticum
; 48.8% identity with the VEB-1 class A β-lactamase from
E. coli
; 40 to 42% identity with CblA of
Bacteroides uniformis
, PER-1 of
Pseudomonas aeruginosa
, and PER-2 of
Salmonella typhimurium
; and 39% identity with CepA of
Bacteroides fragilis
. The partially purified TLA-1 β-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum β-lactamase of Ambler class A.
Susceptibilities of
Yersinia pestis
Strains to 12 Antimicrobial Agents
Authors: Wong Jane D. , Barash Jason R. , Sandfort Rebecca F. , Janda J. Michael
Published: July 2002

Ninety-two strains of
Yersinia pestis
recovered over a 21-year period were evaluated for susceptibility to traditional and newer antimicrobial agents. In vitro resistance was noted only against rifampin and imipenem (∼20% of strains). The most active compounds (MIC at which 90% of the isolates tested are inhibited) against
Y. pestis
were cefixime, ceftriaxone, trimethoprim-sulfamethoxazole, and trovafloxacin.
Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of
Streptococcus pneumoniae
Authors: Blondeau Joseph M. , Zhao Xilin , Hansen Glen , Drlica Karl
Published: July 2002

The mutant prevention concentration (MPC) represents a threshold above which the selective proliferation of resistant mutants is expected to occur only rarely. A provisional MPC (MPC
pr
) was defined and measured for five fluoroquinolones with clinical isolates of
Streptococcus pneumoniae
. Based on their potential for restricting the selection of resistant mutants, the five fluoroquinolones, in descending order, were found to be moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of about 90 clinical isolates that lacked a known resistance mutation had a value of MPC
pr
that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. Since MPC
pr
overestimates MPC, these data identify moxifloxacin and gatifloxacin as good candidates for determining whether MPC
pr
can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of resistance.
Resistance to Moxifloxacin in Toxigenic
Clostridium difficile
Isolates Is Associated with Mutations in
gyrA
Authors: Ackermann Grit , Tang Yajarayma J. , Kueper Robert , Heisig Peter , Rodloff Arne C. , Silva Joseph , Cohen Stuart H.
Published: July 2002

Clostridium difficile
is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of
C. difficile
-associated diarrhea. In this study, we have analyzed 72
C. difficile
isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (
gyrA
), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC ≥ 16 μg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the
gyrA
protein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the
gyrA
gene.
Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases
Authors: Khan Shabana I. , Nimrod Alison C. , Mehrpooya Mohammed , Nitiss John L. , Walker Larry A. , Clark Alice M.
Published: July 2002

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 μg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine,
Saccharomyces cerevisiae
strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.
MET17and Hydrogen Sulfide Formation inSaccharomyces cerevisiae
Authors: Spiropoulos Apostolos , Bisson Linda F.
Published: July 2002

Commercial isolates ofSaccharomyces cerevisiaediffer in the production of hydrogen sulfide (H2S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing theMET17gene, which encodes the bifunctionalO-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H2S release. Overexpression ofMET17resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H2S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H2S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H2S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H2S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H2S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H2S production and release.
Inhibition of Luminescence and Virulence in the Black Tiger Prawn (
Penaeus monodon
) Pathogen
Vibrio harveyi
by Intercellular Signal Antagonists
Authors: Manefield Michael , Harris Lachlan , Rice Scott A. , de Nys Rocky , Kjelleberg Staffan
Published: July 2002

Expression of luminescence in the
Penaeus monodon
pathogen
Vibrio harveyi
is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is
N
-hydroxy butanoyl-
l
-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence in
V. harveyi
are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine alga
Delisea pulchra
on luminescence and toxin production in a virulent strain of
V. harveyi
were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in
V. harveyi
cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from
V. harveyi
cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of
V. harveyi
prawn infections.
Activities of Garlic Oil, Garlic Powder, and Their Diallyl Constituents against
Helicobacter pylori
Authors: O'Gara E. A. , Hill D. J. , Maslin D. J.
Published: July 2002

Chronic
Helicobacter pylori
disease is reduced with
Allium
vegetable intake. This study was designed to assess the in vivo anti-
H. pylori
potential of a variety of garlic substances. The garlic materials all showed substantial but widely differing anti-
H. pylori
effects against all strains and isolates tested. The MICs (range, 8 to 32 μg/ml) and minimum bactericidal concentrations (MBCs) (range, 16 to 32 μg/ml) of undiluted garlic oil (GO) were smaller than those of garlic powder (GP) (MIC range, 250 to 500 μg/ml; MBC range, 250 to 500 μg/ml) but greater than the MIC of allicin (4.0 μg/ml) (Table 2) present in GP. Allicin (MIC, 6 μg/ml; MBC, 6 μg/ml) was more potent than diallyl disulfide (MIC range, 100 to 200 μg/ml; MBC range, 100 to 200 μg/ml), its corresponding sulfide, but of a strength similar to that of diallyl tetrasulfide (MIC range, 3 to 6 μg/ml; MBC range, 3 to 6 μg/ml). Antimicrobial activity of the diallyl sulfides increased with the number of sulfur atoms. Time course viability studies and microscopy showed dose-dependent anti-
H. pylori
effects with undiluted GO, GP, allicin, and diallyl trisulfide after a lag phase of ca. 1 to 2 h. Substantial in vitro anti-
H. pylori
effects of pure GO and GP and their diallyl sulfur components exist, suggesting their potential for in vivo clinical use against
H. pylori
infections.
Detection of
Ralstonia solanacearum
Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay
Authors: Weller S. A. , Elphinstone J. G. , Smith N. C. , Boonham N. , Stead D. E.
Published: July 2002

A fluorogenic (TaqMan) PCR assay was developed to detect
Ralstonia solanacearum
strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of
R. solanacearum
, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of
Taq
DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42
R. solanacearum
and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of
R. solanacearum
to ≥10
2
cells ml
−1
was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of
R. solanacearum
.
Unlabeled Helper Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently Labeled Oligonucleotide Probes
Authors: Fuchs Bernhard M. , Glöckner Frank Oliver , Wulf Jörg , Amann Rudolf
Published: July 2002

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in
Escherichia coli
DSM 30083
T
. The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for
E. coli
. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.
Relative Contributions of Bacteria, Protozoa, and Fungi to In Vitro Degradation of Orchard Grass Cell Walls and Their Interactions
Authors: Lee S. S. , Ha J. K. , Cheng K.-J.
Published: July 2002

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.
Distribution of Oxytetracycline Resistance Plasmids between Aeromonads in Hospital and Aquaculture Environments: Implication of Tn
1721
in Dissemination of the Tetracycline Resistance Determinant Tet A
Authors: Rhodes Glenn , Huys Geert , Swings Jean , Mcgann Patrick , Hiney Maura , Smith Peter , Pickup Roger W.
Published: July 2002

Oxytetracycline-resistant (OT
r
) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OT
r
plasmids from these isolates was investigated. Using
Escherichia coli
J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT
r
plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either
Aeromonas hydrophila
HG3 (eight isolates),
A. veronii
b.v. sobria HG8 (six isolates), and
A. caviae
HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7
tet
gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb
Eco
RI restriction fragment. Hybridization with
inc-rep
probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT
r
plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from
A. salmonicida
strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital
E. coli
strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn
1721
in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different
Aeromonas
species and
E. coli
and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.
Toluene-Degrading Bacteria Are Chemotactic towards the Environmental Pollutants Benzene, Toluene, and Trichloroethylene
Authors: Parales Rebecca E. , Ditty Jayna L. , Harwood Caroline S.
Published: July 2002

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (
Pseudomonas putida
F1,
Ralstonia pickettii
PKO1, and
Burkholderia cepacia
G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene.
Pseudomonas mendocina
KR1 and
P. putida
PaW15 did not show a convincing response. The chemotactic responses of
P. putida
F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for
P. putida
F1, including benzene and ethylbenzene, were chemoattractants.
P. putida
F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by
P. putida
F1. Mutant strains of
P. putida
F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in
P. putida
F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.
Growth of
Pseudomonas mendocina
on Fe(III) (Hydr)Oxides
Authors: Hersman L. E. , Forsythe J. H. , Ticknor L. O. , Maurice P. A.
Published: July 2002

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe,
Pseudomonas mendocina
, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe.
P. mendocina
obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of
P. mendocina
in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.
Variation in Susceptibility to
Bacillus thuringiensis
Toxins among Unselected Strains of
Plutella xylostella
Authors: González-Cabrera J. , Herrero S. , Sayyed A. H. , Escriche B. , Liu Y. B. , Meyer S. K. , Wright D. J. , Tabashnik B. E. , Ferré J.
Published: July 2002

So far, the only insect that has evolved resistance in the field to
Bacillus thuringiensis
toxins is the diamondback moth (
Plutella xylostella
). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to
B. thuringiensis
toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to
B. thuringiensis
toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five
B. thuringiensis
toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.
Role of Intermolecular Disulfide Bonds of the Organic Solvent-Stable PST-01 Protease in Its Organic Solvent Stability
Authors: Ogino Hiroyasu , Uchiho Takeshi , Yokoo Jyunko , Kobayashi Reina , Ichise Rikiya , Ishikawa Haruo
Published: July 2002

The PST-01 protease is secreted by the organic solvent-tolerant microorganism
Pseudomonas aeruginosa
PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.
Functional Analysis of the
Lactococcus lactis galU
and
galE
Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis
Authors: Boels Ingeborg C. , Ramos Ana , Kleerebezem Michiel , de Vos Willem M.
Published: July 2002

We studied the UDP-glucose pyrophosphorylase (
galU
) and UDP-galactose epimerase (
galE
) genes of
Lactococcus lactis
MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in
L. lactis
. The lactococcal
galU
gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The
L. lactis galU
gene could complement an
Escherichia coli galU
mutant, and overexpression of this gene in
L. lactis
under control of the inducible
nisA
promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the
galE
gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the
galE
disruption mutant, while the level of EPS production was approximately one-half the wild-type level.
Spatial and Resource Factors Influencing High Microbial Diversity in Soil
Authors: Zhou Jizhong , Xia Beicheng , Treves David S. , Wu L.-Y. , Marsh Terry L. , O’Neill Robert V. , Palumbo Anthony V. , Tiedje James M.
Published: July 2002

To begin defining the key determinants that drive microbial community structure in soil, we examined 29 soil samples from four geographically distinct locations taken from the surface, vadose zone, and saturated subsurface using a small-subunit rRNA-based cloning approach. While microbial communities in low-carbon, saturated, subsurface soils showed dominance, microbial communities in low-carbon surface soils showed remarkably uniform distributions, and all species were equally abundant. Two diversity indices, the reciprocal of Simpson’s index (1/
D
) and the log series index, effectively distinguished between the dominant and uniform diversity patterns. For example, the uniform profiles characteristic of the surface communities had diversity index values that were 2 to 3 orders of magnitude greater than those for the high-dominance, saturated, subsurface communities. In a site richer in organic carbon, microbial communities consistently exhibited the uniform distribution pattern regardless of soil water content and depth. The uniform distribution implies that competition does not shape the structure of these microbial communities. Theoretical studies based on mathematical modeling suggested that spatial isolation could limit competition in surface soils, thereby supporting the high diversity and a uniform community structure. Carbon resource heterogeneity may explain the uniform diversity patterns observed in the high-carbon samples even in the saturated zone. Very high levels of chromium contamination (e.g., >20%) in the high-organic-matter soils did not greatly reduce the diversity. Understanding mechanisms that may control community structure, such as spatial isolation, has important implications for preservation of biodiversity, management of microbial communities for bioremediation, biocontrol of root diseases, and improved soil fertility.
Resolution of
Prochlorococcus
and
Synechococcus
Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences
Authors: Rocap Gabrielle , Distel Daniel L. , Waterbury John B. , Chisholm Sallie W.
Published: July 2002

Cultured isolates of the marine cyanobacteria
Prochlorococcus
and
Synechococcus
vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32
Prochlorococcus
isolates and 25
Synechococcus
isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among
Prochlorococcus
strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of
Prochlorococcus
into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A
Synechococcus
into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of
Prochlorococcus
and
Synechococcus
consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.
Role of
Bacillus thuringiensis
Cry1 δ Endotoxin Binding in Determining Potency during Lepidopteran Larval Development
Authors: Gilliland Androulla , Chambers Catherine E. , Bone Eileen J. , Ellar David J.
Published: July 2002

Five economically important crop pests,
Manduca sexta
,
Pieris brassicae
,
Mamestra brassicae
,
Spodoptera exigua
, and
Agrotis ipsilon
, were tested at two stages of larval development for susceptibility to
Bacillus thuringiensis
toxins Cry1Ac, Cry1Ca, Cry1J, and Cry1Ba. Bioassay results for
M. sexta
showed that resistance to all four Cry toxins increased from the neonate stage to the third-instar stage; the increase in resistance was most dramatic for Cry1Ac, the potency of which decreased 37-fold. More subtle increases in resistance during larval development were seen in
M. brassicae
for Cry1Ca and in
P. brassicae
for Cry1Ac and Cry1J. By contrast, the sensitivity of
S. exigua
did not change during development. At both larval stages,
A. ipsilon
was resistant to all four toxins. Because aminopeptidase N (APN) is a putative Cry1 toxin binding protein, APN activity was measured in neonate and third-instar brush border membrane vesicles (BBMV). With the exception of
S. exigua
, APN activity was found to be significantly lower in neonates than in third-instar larvae and thus inversely correlated with increased resistance during larval development. The binding characteristics of iodinated Cry1 toxins were determined for neonate and third-instar BBMV. In
M. sexta
, the increased resistance to Cry1Ac and Cry1Ba during larval development was positively correlated with fewer binding sites in third-instar BBMV than in neonate BBMV. The other species-instar-toxin combinations did not reveal positive correlations between potency and binding characteristics. The correlation between binding and potency was inconsistent for the species-instar-toxin combinations used in this study, reaffirming the complex mode of action of Cry1 toxins.
Improved Culturability of Soil Bacteria and Isolation in Pure Culture of Novel Members of the Divisions
Acidobacteria
,
Actinobacteria
,
Proteobacteria
, and
Verrucomicrobia
Authors: Janssen Peter H. , Yates Penelope S. , Grinton Bronwyn E. , Taylor Paul M. , Sait Michelle
Published: July 2002

The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions
Actinobacteria
,
Acidobacteria
,
Proteobacteria
, and
Verrucomicrobia
.
Prevalence of
Enterocytozoon bieneusi
in Swine: an 18-Month Survey at a Slaughterhouse in Massachusetts
Authors: Buckholt Michael A. , Lee John H. , Tzipori Saul
Published: July 2002

Slaughterhouse pig samples were analyzed by PCR for
Enterocytozoon bieneusi
infection. Thirty-two percent were found to be positive, with rates being higher over the summer months. Three isolates from pigs were identical in their ribosomal internal transcribed spacer sequence to human
E. bieneusi
type D, two were identical to type F (from a pig), and nine were previously unreported. The viability of these spores was demonstrated by their ability to infect gnotobiotic piglets. The presence of the infection in liver was shown by in situ hybridization.
Novel Approach for Specific Detection of Herpes Simplex Virus Type 1 and 2 Antibodies and Immunoglobulin G and M Antibodies
Authors: Ohana Bella , Lipson Mark , Vered Noga , Srugo Isaac , Ahdut Moshe , Morag Abraham
Published: July 2002

Recently a few new herpes simplex virus (HSV) type-specific serological diagnostic tests have been introduced to the commercial market, but these tests have some limitations. Moreover, it is not yet clear which commercial test can be regarded as a “gold standard” for the serodiagnosis of HSV infections. In order to improve the clinical diagnostic value of serological tests for the detection of HSV infections, we developed novel, competition-based enzyme-linked immunosorbent assays for the specific determination of HSV type 2 antibodies (SeroHSV2) and HSV type 1 antibodies (SeroHSV1) and two complementary tests for the detection of HSV immunoglobulin M (IgM) and IgG antibodies (SeroHSV IgM and SeroHSV IgG). These four new kits were evaluated in comparison with some commercial kits for the detection of HSV antibodies that are commonly used at present in Israeli clinical laboratories. The results indicate that SeroHSV2 is highly sensitive (>92%) and highly specific (>94%). SeroHSV2 does not cross-react with other alphaherpesvirus antibodies. SeroHSV1 is highly sensitive (>94%) and specific (>91%) compared to four commercial available kits. SeroHSV IgM is highly specific (>92%) in comparison with other commercial HSV IgM tests. The sensitivity of SeroHSV IgM ranges between 50 and 70% compared to these tests. Further investigation of the discrepant results obtained by using in-house competition tests indicated that SeroHSV IgM is more sensitive. SeroHSV IgG was also found to be highly sensitive (>94%) and highly specific (>92%) compared to the other commercial HSV IgG tests.
Antibody Responses to MAP 1B and Other
Cowdria ruminantium
Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater
Authors: Semu S. M. , Peter T. F. , Mukwedeya D. , Barbet A. F. , Jongejan F. , Mahan S. M.
Published: July 2002

Serological diagnosis of heartwater or
Cowdria ruminantium
infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted
C. ruminantium
infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with
C. ruminantium
via
Amblyomma hebraeum
ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other
C. ruminantium
antigens are down regulated in cattle despite repeated exposure to
C. ruminantium
via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of
C. ruminantium
exposure in cattle in areas of endemic heartwater infection.
Vγ9Vδ2 T Cells in Human Legionellosis
Authors: Kroca Michal , Johansson Anders , Sjöstedt Anders , Tärnvik Arne
Published: July 2002

In humans, expansion of circulating Vγ9Vδ2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to γδ T cells. Levels of Vγ9Vδ2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused byLegionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (± the standard deviation) of Vγ9Vδ2+T cells among CD3+cells was 1.0% ± 0.5%, compared to 5.0% ± 3.9% in healthy control subjects (P< 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as ≈15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of γδ+T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P< 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P< 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vγ9Vδ2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vγ9Vδ2 T cells. Possibly, Vγ9Vδ2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.