bluuebunny/crossref_metadata_2025 huggingface dataset:
Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of
Pseudomonas aeruginosa
Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance
Authors: Akasaka Takaaki , Tanaka Mayumi , Yamaguchi Akihito , Sato Kenichi
Published: July 2002

The major mechanism of resistance to fluoroquinolones for
Pseudomonas aeruginosa
is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) of
gyrA, gyrB, parC
, and
parE
genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in
parC
or
parE
which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in
gyrA
and
parC
, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.
Novel Synthetic Polyamines Are Effective in the Treatment of Experimental Microsporidiosis, an Opportunistic AIDS-Associated Infection
Authors: Bacchi Cyrus J. , Weiss Louis M. , Lane Schenella , Frydman Benjamin , Valasinas Aldonia , Reddy Venodhar , Sun Jerry S. , Marton Laurence J. , Khan Imitiaz A. , Moretto Magali , Yarlett Nigel , Wittner Murray
Published: July 2002

Microsporidia are eukaryotic obligate intracellular protists that are emerging pathogens in immunocompromised hosts, such as patients with AIDS or patients who have undergone organ transplantation. We have demonstrated in vitro and in vivo that synthetic polyamine analogs are effective antimicrosporidial agents with a broad therapeutic window. CD8-knockout mice or nude mice infected with the microsporidianEncephalitozoon cuniculiwere cured when they were treated with four different novel polyamine analogs at doses ranging from 1.25 to 5 mg/kg of body weight/day for a total of 10 days. Cured animals demonstrated no evidence of parasitemia by either PCR or histologic staining of tissues 30 days after untreated control animals died.
Lack of Effect of Simultaneously Administered Didanosine Encapsulated Enteric Bead Formulation (Videx EC) on Oral Absorption of Indinavir, Ketoconazole, or Ciprofloxacin
Authors: Damle Bharat D. , Mummaneni Vanaja , Kaul Sanjeev , Knupp Catherine
Published: July 2002

Didanosine formulation that contains a buffer to prevent it from acid-mediated degradation can result in a significant decrease in the oral absorption of certain drugs because of interactions with antacids. An enteric formulation of didanosine is unlikely to cause such drug interactions because it lacks antacids. This study was undertaken to determine whether the enteric bead formulation of didanosine (Videx EC) influences the bioavailability of indinavir, ketoconazole, and ciprofloxacin, three drugs that are representative of a broader class of drugs affected by interaction with antacids. Healthy subjects of either gender were enrolled in three separate open-label, single-dose, two-way crossover studies. Subjects were randomized to treatment A (800 mg of indinavir, 200 mg of ketoconazole, or 750 mg of ciprofloxacin) or treatment B (same dose of indinavir, ketoconazole, or ciprofloxacin, but with 400 mg of didanosine as an encapsulated enteric bead formulation). A lack of interaction was concluded if the 90% confidence interval (CI) of the ratio of the geometric means of log-transformedCmaxand AUC0-∞values (i.e., values for the area under the concentration-time curve from time zero to infinity) of indinavir, ketoconazole, and ciprofloxacin were contained entirely between 0.75 and 1.33. For indinavir (n= 23), the point estimate (90% CI; minimum, maximum) of the ratios ofCmaxand AUC0-∞values were 0.99 (0.91, 1.06) and 0.96 (0.91, 1.02), respectively. In the ketoconazole study, 3 of 24 subjects showed anomalous absorption of ketoconazole (i.e., an ∼8-fold-lower AUC compared to historical data), which was the reference treatment. A post hoc analysis performed after these three subjects were excluded indicated that the point estimates (90% CI) of the ratios of Cmaxand AUC0-∞values were 0.99 (0.86, 1.14) and 0.97 (0.85, 1.10), respectively. For ciprofloxacin (n= 16), the point estimate (90% CI) of the ratios ofCmaxand AUC0-∞values were 0.92 (0.79, 1.07) and 0.91 (0.76, 1.08), respectively. All three studies clearly indicated a lack of interaction. TheTmaxandt1/2for indinavir, ketoconazole, and ciprofloxacin were similar between treatments. Our results showed that the lack of interaction of didanosine encapsulated enteric bead formulation with indinavir, ketoconazole, and ciprofloxacin indicates that this enteric formulation of didanosine can be concomitantly administered with drugs whose bioavailability is known to be reduced by interaction with antacids.
Selection of
Streptococcus pneumoniae
Mutants Having Reduced Susceptibility to Moxifloxacin and Levofloxacin
Authors: Li Xinying , Zhao Xilin , Drlica Karl
Published: July 2002

With
Streptococcus pneumoniae
, moxifloxacin was 4- and 10-fold more effective than levofloxacin at restricting selection of resistant mutants and at killing resistant mutants, respectively. The selection frequency for first-step topoisomerase mutants was 1,000 times lower for moxifloxacin than for levofloxacin; this difference was lost when second-step mutants were selected.
mar
Operon Involved in Multidrug Resistance of
Enterobacter aerogenes
Authors: Chollet Renaud , Bollet Claude , Chevalier Jacqueline , Malléa Monique , Pagès Jean-Marie , Davin-Regli Anne
Published: July 2002

We determined the sequence of the entire
marRAB
operon in
Enterobacter aerogenes
. It is functionally and structurally analogous to the
Escherichia coli
operon. The overexpression of
E. aerogenes
MarA induces a multidrug resistance phenotype in a susceptible strain, demonstrated by a noticeable resistance to various antibiotics, a decrease in immunodetected porins, and active efflux of norfloxacin.
Inhibitory Activities of Quinolones against DNA Gyrase and Topoisomerase IV of
Enterococcus faecalis
Authors: Onodera Yoshikuni , Okuda Jun , Tanaka Mayumi , Sato Kenichi
Published: July 2002

We have cloned the DNA gyrase and topoisomerase IV genes of
Enterococcus faecalis
to examine the actions of quinolones against
E. faecalis
genetically and enzymatically. We first generated levofloxacin-resistant mutants of
E. faecalis
by stepwise selection with increasing drug concentrations and analyzed the quinolone resistance-determining regions of
gyrA
and
parC
from the resistant mutants. Isogenic mutants with low-level resistance contained a mutation in
gyrA
, whereas those with higher levels of resistance had mutations in both
gyrA
and
parC
. These results suggested that
gyrA
is the primary target for levofloxacin in
E. faecalis
. We then purified the recombinant DNA gyrase and topoisomerase IV enzymes of
E. faecalis
and measured the in vitro inhibitory activities of quinolones against these enzymes. The 50% inhibitory concentrations (IC
50
s) of levofloxacin, ciprofloxacin, sparfloxacin, tosufloxacin, and gatifloxacin for DNA gyrase were found to be higher than those for topoisomerase IV. In conflict with the genetic data, these results indicated that topoisomerase IV would be the primary target for quinolones in
E. faecalis
. Among the quinolones tested, the IC
50
of sitafloxacin (DU-6859a), which shows the greatest potency against enterococci, for DNA gyrase was almost equal to that for topoisomerase IV; its IC
50
s were the lowest among those of all the quinolones tested. These results indicated that other factors can modulate the effect of target affinity to determine the bacterial killing pathway, but the highest inhibitory actions against both enzymes correlated with good antienterococcal activities.
Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds
Authors: Cowan Stacie E. , Gilbert Eric , Liepmann Dorian , Keasling J. D.
Published: July 2002

There is limited knowledge of interspecies interactions in biofilm communities. In this study,
Pseudomonas
sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and
Pseudomonas putida
DMP1, a
p
-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various
p
-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of
p
-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the
p
-cresol concentrations increased, and biofilm formation was severely inhibited at high
p
-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all
p
-cresol concentrations, although at high
p
-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and
Escherichia coli
ATCC 33456, an adhesive strain that does not mineralize
p
-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.
Cometabolic Degradation of Dibenzofuran by Biphenyl-Cultivated
Ralstonia
sp. Strain SBUG 290
Authors: Becher Dörte , Specht Michael , Hammer Elke , Francke Wittko , Schauer Frieder
Published: July 2002

Cells of the gram-negative bacterium
Ralstonia
sp. strain SBUG 290 grown in the presence of biphenyl are able to cooxidize dibenzofuran which has been 1,2-hydroxylated. Meta cleavage of the 1,2-dihydroxydibenzofuran between carbon atoms 1 and 9b produced 2-hydroxy-4-(3′-oxo-3′
H
-benzofuran-2′-yliden)but-2-enoic acid, which was degraded completely via salicylic acid. The presence of these intermediates indicates a degradation mechanism for dibenzofuran via lateral dioxygenation by
Ralstonia
sp. strain SBUG 290.
Transition from Anaerobic to Aerobic Growth Conditions for the Sulfate-Reducing Bacterium
Desulfovibrio oxyclinae
Results in Flocculation
Authors: Sigalevich Pavel , Meshorer Eran , Helman Yael , Cohen Yehuda
Published: July 2002

A chemostat culture of the sulfate-reducing bacterium
Desulfovibrio oxyclinae
isolated from the oxic layer of a hypersaline cyanobacterial mat was grown anaerobically and then subjected to gassing with 1% oxygen, both at a dilution rate of 0.05 h
−1
. The sulfate reduction rate under anaerobic conditions was 370 nmol of SO
4
2−
mg of protein
−1
min
−1
. At the onset of aerobic gassing, sulfate reduction decreased by 40%, although viable cell numbers did not decrease. After 42 h, the sulfate reduction rate returned to the level observed in the anaerobic culture. At this stage the growth yield increased by 180% compared to the anaerobic culture to 4.4 g of protein per mol of sulfate reduced. Protein content per cell increased at the same time by 40%. The oxygen consumption rate per milligram of protein measured in washed cell suspensions increased by 80%, and the thiosulfate reduction rate of the same samples increased by 29% with lactate as the electron donor. These findings indicated possible oxygen-dependent enhancement of growth. After 140 h of growth under oxygen flux, formation of cell aggregates 0.1 to 3 mm in diameter was observed. Micrometer-sized aggregates were found to form earlier, during the first hours of exposure to oxygen. The respiration rate of
D. oxyclinae
was sufficient to create anoxia inside clumps larger than 3 μm, while the levels of dissolved oxygen in the growth vessel were 0.7 ± 0.5 μM. Aggregation of sulfate-reducing bacteria was observed within a
Microcoleus chthonoplastes
-dominated layer of a cyanobacterial mat under daily exposure to oxygen concentrations of up to 900 μM.
Desulfonema
-like sulfate-reducing bacteria were also common in this environment along with other nonaggregated sulfate-reducing bacteria. Two-dimensional mapping of sulfate reduction showed heterogeneity of sulfate reduction activity in this oxic zone.
Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes
Authors: Takai Ken , Horikoshi Koki
Published: July 2002

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.
Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of
Lactobacillus helveticus
CRL 1062
Authors: Hebert Elvira M. , Raya Raul R. , De Giori Graciela S.
Published: July 2002

The nutritional requirements of
Lactobacillus helveticus
CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of
L. helveticus
CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or β-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%.
Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates
Authors: Ramos Cayo , Mølbak Lars , Molin Søren
Published: July 2002

The growth activity of
Pseudomonas putida
cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal
Escherichia coli rrnB
P1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a
P. putida
strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h
−1
emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in
P. putida
cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of
P. putida
cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems.
Distribution of
Nitrosomonas europaea
and
Paracoccus denitrificans
Immobilized in Tubular Polymeric Gel for Nitrogen Removal
Authors: Uemoto Hiroaki , Saiki Hiroshi
Published: July 2002

To improve the cooperative removal of nitrogen by
Nitrosomonas europaea
and
Paracoccus denitrificans
, we controlled their distribution in a tubular gel. When ethanol was supplied inside the tubular gel as an electron donor, their distributions overlapped in the external region of the gel. By changing the electron donor from ethanol to gaseous hydrogen, the distribution of
P. denitrificans
shifted to the inside of the tube and was separated from that of
N. europaea
. The separation resulted in an increase of the oxidation rate of ammonia by 25%.
Cloning and Sequencing of an Alkaline Protease Gene from
Bacillus lentus
and Amplification of the Gene on the
B. lentus
Chromosome by an Improved Technique
Authors: Jørgensen Per Linå , Tangney Martin , Pedersen Poul Erik , Hastrup Sven , Diderichsen Børge , Jørgensen Steen T.
Published: July 2002

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.
Atypical Polyphosphate Accumulation by the Denitrifying Bacterium
Paracoccus denitrificans
Authors: Barak Yoram , van Rijn Jaap
Published: July 2002

Polyphosphate accumulation by
Paracoccus denitrificans
was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that
P. denitrificans
is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.
Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by
Yarrowia lipolytica
Authors: Waché Yves , Laroche Céline , Bergmark Karin , Møller-Andersen Charlotte , Aguedo Mario , Le Dall Marie-Thérèse , Wang Huijie , Nicaud Jean-Marc , Belin Jean-Marc
Published: July 2002

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast
Yarrowia lipolytica
by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (
POX1
through
POX5
) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δ
pox3
, which produced 220 mg of γ-decalactone per liter after 24 h. The Δ
pox2 Δpox3
double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δ
pox2 Δpox3 Δpox5
triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δ
pox2 Δpox3 Δpox4 Δpox5
multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.
Influence of
Acanthamoeba castellanii
on Intracellular Growth of Different
Legionella
Species in Human Monocytes
Authors: Neumeister B. , Reiff G. , Faigle M. , Dietz K. , Northoff H. , Lang F.
Published: July 2002

Previous studies using a murine model of coinhalation of
Legionella pneumophila
and
Hartmannella vermiformis
have shown a significantly enhanced intrapulmonary growth of
L. pneumophila
in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449–2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and
Acanthamoeba castellanii
, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas
L. pneumophila
has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with
Acanthamoeba castellanii
, significantly enhanced replication of
L. gormanii
,
L. micdadei
,
L. steigerwaltii
,
L. longbeachae
, and
L. dumoffii
was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with
Legionella
-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected
A. castellanii
and
Legionella
-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.
Natural Assemblages of Marine Proteobacteria and Members of the
Cytophaga-Flavobacter
Cluster Consuming Low- and High-Molecular-Weight Dissolved Organic Matter
Authors: Cottrell Matthew T. , Kirchman David L.
Published: July 2002

We used a method that combines microautoradiography with hybridization of fluorescent rRNA-targeted oligonucleotide probes to whole cells (MICRO-FISH) to test the hypothesis that the relative contributions of various phylogenetic groups to the utilization of dissolved organic matter (DOM) depend solely on their relative abundance in the bacterial community. We found that utilization of even simple low-molecular-weight DOM components by bacteria differed across the major phylogenetic groups and often did not correlate with the relative abundance of these bacterial groups in estuarine and coastal environments. The
Cytophaga-Flavobacter
cluster was overrepresented in the portion of the assemblage consuming chitin,
N
-acetylglucosamine, and protein but was generally underrepresented in the assemblage consuming amino acids. The amino acid-consuming assemblage was usually dominated by the α subclass of the class
Proteobacteria
, although the representation of α-proteobacteria in the protein-consuming assemblages was about that expected from their relative abundance in the entire bacterial community. In our experiments, no phylogenetic group dominated the consumption of all DOM, suggesting that the participation of a diverse assemblage of bacteria is essential for the complete degradation of complex DOM in the oceans. These results also suggest that the role of aerobic heterotrophic bacteria in carbon cycling would be more accurately described by using three groups instead of the single bacterial compartment currently used in biogeochemical models.
Chlorine, Chloramine, Chlorine Dioxide, and Ozone Susceptibility of
Mycobacterium avium
Authors: Taylor Robert H. , Falkinham Joseph O. , Norton Cheryl D. , LeChevallier Mark W.
Published: July 2002

Environmental and patient isolates of
Mycobacterium avium
were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five
M. avium
strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of
M. avium
in drinking water.
Anaerobic-Aerobic Process for Microbial Degradation of Tetrabromobisphenol A
Authors: Ronen Zeev , Abeliovich Aharon
Published: July 2002

Tetrabromobisphenol A (TBBPA) is a flame retardant that is used as an additive during manufacturing of plastic polymers and electronic circuit boards. Little is known about the fate of this compound in the environment. In the current study we investigated biodegradation of TBBPA, as well as 2,4,6-tribromophenol (TBP), in slurry of anaerobic sediment from a wet ephemeral desert stream bed contaminated with chemical industry waste. Anaerobic incubation of the sediment with TBBPA and peptone-tryptone-glucose-yeast extract medium resulted in a 80% decrease in the TBBPA concentration and accumulation of a single metabolite. This metabolite was identified by gas chromatography-mass spectrometry (GC-MS) as nonbrominated bisphenol A (BPA). On the other hand, TBP was reductively dehalogenated to phenol, which was further metabolized under anaerobic conditions. BPA persisted in the anaerobic slurry but was degraded aerobically. A gram-negative bacterium (strain WH1) was isolated from the contaminated soil, and under aerobic conditions this organism could use BPA as a sole carbon and energy source. During degradation of BPA two metabolites were detected in the culture medium, and these metabolites were identified by GC-MS and high-performance liquid chromatography as 4-hydroxybenzoic acid and 4-hydroxyacetophenone. Both of those compounds were utilized by WH1 as carbon and energy sources. Our findings demonstrate that it may be possible to use a sequential anaerobic-aerobic process to completely degrade TBBPA in contaminated soils.
Comparative Survival Rates of Human-Derived Probiotic
Lactobacillus paracasei
and
L. salivarius
Strains during Heat Treatment and Spray Drying
Authors: Gardiner G. E. , O'Sullivan E. , Kelly J. , Auty M. A. E. , Fitzgerald G. F. , Collins J. K. , Ross R. P. , Stanton C.
Published: July 2002

Spray drying of skim milk was evaluated as a means of preserving
Lactobacillus paracasei
NFBC 338 and
Lactobacillus salivarius
UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 10
9
CFU/g for NFBC 338 and 5.2 × 10
7
CFU/g for UCC 118. Thus,
L. paracasei
NFBC 338 survived better than
L. salivarius
UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD
Bac
Light viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ∼1 × 10
9
CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 10
7
to 9.5 × 10
6
CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both
Lactobacillus
strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.
Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates
Authors: Duetz Wouter A. , Rüedi Lorenz , Hermann Robert , O'Connor Kevin , Büchs Jochen , Witholt Bernard
Published: July 2002

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of
Pseudomonas putida
and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter
−1
h
−1
, corresponding to a mass transfer coefficient of 188 h
−1
) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed
P. putida
to reach a cell density of approximately 9 g (dry weight) liter
−1
during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera
Pseudomonas
,
Rhodococcus
, and
Alcaligenes
, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.
Relationship between Membrane Damage and Cell Death in Pressure-Treated
Escherichia coli
Cells: Differences between Exponential- and Stationary-Phase Cells and Variation among Strains
Authors: Pagán Rafael , Mackey Bernard
Published: July 2002

The relationship between membrane damage and loss of viability following pressure treatment was examined in
Escherichia coli
strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-β-
d
-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.
Extremely Halophilic
Bacteria
in Crystallizer Ponds from Solar Salterns
Authors: Antón Josefa , Rosselló-Mora Ramón , Rodríguez-Valera Francisco , Amann Rudolf
Published: July 2002

It is generally assumed that hypersaline environments with sodium chloride concentrations close to saturation are dominated by halophilic members of the domain
Archaea
, while
Bacteria
are not considered to be relevant in this kind of environment. Here, we report the high abundance and growth of a new group of hitherto-uncultured
Bacteria
in crystallizer ponds (salinity, from 30 to 37%) from multipond solar salterns. In the present study, these
Bacteria
constituted from 5 to 25% of the total prokaryotic community and were affiliated with the
Cytophaga-Flavobacterium-Bacteroides
phylum. Growth was demonstrated in saturated NaCl. A provisional classification of this new bacterial group as “
Candidatus
Salinibacter gen. nov.” is proposed. The perception that
Archaea
are the only ecologically relevant prokaryotes in hypersaline aquatic environments should be revised.
Factors Affecting Exocellular Polysaccharide Production byLactobacillus delbrueckiisubsp.bulgaricusGrown in a Chemically Defined Medium
Authors: Petry Sandrine , Furlan Sylviane , Crepeau Marie-Jeanne , Cerning Jutta , Desmazeaud Michel
Published: July 2002

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains ofLactobacillus delbrueckiisubsp.bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strainL. delbrueckiisubsp.bulgaricusCNRZ 1187 contained galactose and glucose, and that of strainL. delbrueckiisubsp.bulgaricusCNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions,L. delbrueckiisubsp.bulgaricusstrains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.
Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps
Authors: Chandler Darrell P. , Stults Jennie R. , Cebula Sharon , Schuck Beatrice L. , Weaver Derek W. , Anderson Kevin K. , Egholm Michael , Brockman Fred J.
Published: July 2002

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO
4
, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO
4
, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10
−21
M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.
Characteristics of
Vibrio parahaemolyticus
O3:K6 from Asia
Authors: Wong Hin-Chung , Liu Shu-Hui , Wang Tien-Kuei , Lee Chih-Lung , Chiou Chien-Shun , Liu Ding-Ping , Nishibuchi Mitsuaki , Lee Bok-Kwon
Published: July 2002

A variety of serovars of the food-borne pathogen
Vibrio parahaemolyticus
normally cause infection. Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan. For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics. After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups. The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns. Pattern I1 was the major one for strains from Japan, Korea, and Taiwan. All recently isolated O3:K6 strains carried the thermostable direct hemolysin (
tdh
) gene. No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses. Results in this study confirmed that the recently isolated O3:K6 strains of
V. parahaemolyticus
are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains.
Molecular Phylogeny of
Anabaena circinalis
and Its Identification in Environmental Samples by PCR
Authors: Fergusson Kim M. , Saint Christopher P.
Published: July 2002

Although the cyanobacterium
Anabaena circinalis
occurs worldwide, Australian isolates are believed to exclusively possess the saxitoxin group neurotoxins (paralytic shellfish poisons). Identification of
A. circinalis
in a mixed population is complicated due to limited morphological differences between
Anabaena
species. Sequence analysis of the DNA-dependent RNA polymerase (
rpoC1
) gene from 24
Anabaena
isolates, including 12 designated
A. circinalis
, permitted a phylogenetic analysis to be performed. In addition, an
A. circinalis
-specific PCR was developed and tested successfully on environmental samples.
Analysis of Bacterial Communities in the Rhizosphere of Chrysanthemum via Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA as Well as DNA Fragments Coding for 16S rRNA
Authors: Duineveld Bernadette M. , Kowalchuk George A. , Keijzer Anneke , van Elsas Jan Dirk , van Veen Johannes A.
Published: July 2002

The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as
Pseudomonas
,
Comamonas
,
Variovorax
, and
Acetobacter
, or typical of root-free soil environments, such as
Bacillus
and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.
Molecular Ecology of Tetracycline Resistance: Development and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins
Authors: Aminov R. I. , Garrigues-Jeanjean N. , Mackie R. I.
Published: July 2002

Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by
tet
and
otrA
, consisted of genes from the antibiotic-producing organisms
Streptomyces rimosus
and
Streptomyces lividans
. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except
otrA
and
tet
, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The
tet
(O) and
tet
(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the
tet
(O) gene, and 22% of them also carried
tet
(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals.
Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts
Authors: Higgs Richard E. , Zahn James A. , Gygi Jeffrey D. , Hilton Matthew D.
Published: July 2002

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.