bluuebunny/crossref_metadata_2025 huggingface dataset:
Potato Dextrose Agar Antifungal Susceptibility Testing for Yeasts and Molds: Evaluation of Phosphate Effect on Antifungal Activity of CMT-3
Authors: Liu Yu , Tortora George , Ryan Maria E. , Lee Hsi-Ming , Golub Lorne M.
Published: July 2002

The broth macrodilution method (BMM) for antifungal susceptibility testing, approved by the National Committee for Clinical Laboratory Standards (NCCLS), was found to have deficiencies in testing of the antifungal activity of a new type of antifungal agent, a nonantibacterial chemically modified tetracycline (CMT-3). The high content of phosphate in the medium was found to greatly increase the MICs of CMT-3. To avoid the interference of phosphate in the test, a new method using potato dextrose agar (PDA) as a culture medium was developed. Eight strains of fungi, including five American Type Culture Collection strains and three clinical isolates, were used to determine the MICs of amphotericin B and itraconazole with both the BMM and the PDA methods. The MICs of the two antifungal agents determined with the PDA method showed 99% agreement with those determined with the BMM method within 1 log
2
dilution. Similarly, the overall reproducibility of the MICs with the PDA method was above 97%. Three other antifungal agents, fluconazole, ketoconazole, and CMT-3, were also tested in parallel against yeasts and molds with both the BMM and the PDA methods. The MICs of fluconazole and ketoconazole determined with the PDA method showed 100% agreement within 1 log
2
dilution of those obtained with the BMM method. However, the MICs of CMT-3 determined with the BMM method were as high as 128 times those determined with the PDA method. The effect of phosphate on the antifungal activity of CMT-3 was evaluated by adding Na
2
HPO
4
to PDA in the new method. It was found that the MIC of CMT-3 against a
Penicillium
sp. increased from 0.5 μg/ml (control) to 2.0 μg/ml when the added phosphate was used at a concentration of 0.8 mg/ml, indicating a strong interference of Na
2
HPO
4
with the antifungal activity of CMT-3. Except for fluconazole, all the other antifungal agents demonstrated clear end points among the yeasts and molds tested. Nevertheless, with its high reproducibility, good agreement with NCCLS proposed MIC ranges, and lack of interference of phosphate, the PDA method shows promise as a useful assay for antifungal susceptibility testing and screening for new antifungal agents, especially for drugs that may be affected by high (supraphysiologic) phosphate concentrations.
Antimicrobial Activity of a Novel Catheter Lock Solution
Authors: Shah Chirag B. , Mittelman Marc W. , Costerton J. W. , Parenteau Stephen , Pelak Michael , Arsenault Richard , Mermel Leonard A.
Published: July 2002

Intravascular catheter-associated bloodstream infections significantly increase rates of morbidity and hospital costs. Microbial colonization and development of biofilms, which are known to be recalcitrant to antibiotic therapy, often lead to the loss of otherwise patent vascular access systems. We evaluated a new taurolidine- and citrate-based catheter lock solution (Neutrolin; Biolink Corporation, Norwell, Mass.) for its activity against planktonic microbes, antimicrobial activity in a catheter model, and biofilm eradication activity. In studies of planktonic microbes, after 24 h of contact, 675 mg of taurolidine-citrate solution per liter caused >99% reductions in the initial counts of
Staphylococcus aureus
,
Staphylococcus epidermidis
,
Pseudomonas aeruginosa
, and
Entercoccus faecalis.
A solution of 13,500 mg/liter was cidal for
Candida albicans
. Ports and attached catheters inoculated with 50 to 600 CFU of these bloodstream isolates per ml were locked with heparin or the taurolidine-citrate solution. After 72 h, there was no growth in the taurolidine-citrate-treated devices but the heparin-treated devices exhibited growth in the range of 6 × 10
2
to 5 × 10
6
CFU/ml. Biofilms were developed on silicone disks in modified Robbins devices with broth containing 6% serum (initial counts, 10
6
to 10
8
CFU/cm
2
). The axenic biofilms were treated for 24 h with taurolidine-citrate or heparin. Taurolidine-citrate exposure resulted in a median reduction of 4.8 logs, whereas heparin treatment resulted in a median reduction of 1.7 logs (
P
< 0.01). No significant differences in the effects of the two treatments against
P. aeruginosa
and
C. albicans
were observed. These findings suggest that taurolidine-citrate is a promising combination agent for the prevention and treatment of intravascular catheter-related infections.
Antibacterial Activities and Characterization of Novel Inhibitors of LpxC
Authors: Clements John M. , Coignard Fanny , Johnson Ian , Chandler Stephen , Palan Shilpa , Waller Andrew , Wijkmans Jac , Hunter Michael G.
Published: July 2002

Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria. Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria. UDP-3-
O
-(
R
-3-hydroxymyristoyl)-
N
-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A. Inhibitors of LpxC have previously been shown to have antibiotic activities. We have screened a metalloenzyme inhibitor library for antibacterial activities against an
Escherichia coli
strain with reduced LpxC activity. From this screen, a series of sulfonamide derivatives of the α-(
R
)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay. Leads from this series showed gram-negative selective activities against members of the
Enterobacteriaceae
,
Serratia marcescens
,
Morganella morganii
,
Haemophilus influenzae
,
Moraxella catarrhalis
, and
Burkholderia cepacia
. BB-78484 was bactericidal against
E. coli
, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis.
E. coli
mutants with decreased susceptibility to BB-78484 were selected. Analysis of these mutants revealed that resistance arose as a consequence of mutations in the
fabZ
or
lpxC
genes. These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials.
PCR-Based Identification of
MAT-1
and
MAT-2
in the
Gibberella fujikuroi
Species Complex
Authors: Steenkamp Emma T. , Wingfield Brenda D. , Coutinho Teresa A. , Zeller Kurt A. , Wingfield Michael J. , Marasas Walter F. O. , Leslie John F.
Published: July 2002

All sexually fertile strains in the
Gibberella fujikuroi
species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs
MAT-1
and
MAT-2
. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an ∼800-bp fragment from
MAT-2
or an ∼200-bp fragment from
MAT-1
is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of
Fusarium moniliforme
(
Fusarium verticillioides
). Neither of the primer pairs amplify fragments from
Fusarium
species such as
Fusarium graminearum
,
Fusarium pseudograminearum
, and
Fusarium culmorum
, which have, or are expected to have,
Gibberella
sexual stages but are thought to be relatively distant from the species in the
G. fujikuroi
species complex. Our results suggest that
MAT
allele sequences are useful indicators of phylogenetic relatedness in these and other
Fusarium
species.
Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments
Authors: Fuller Mark E. , Streger Sheryl H. , Rothmel Randi K. , Mailloux Brian J. , Hall James A. , Onstott Tullis C. , Fredrickson James K. , Balkwill David L. , DeFlaun Mary F.
Published: July 2002

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate,
Comamonas
sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained
Comamonas
sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10
5
CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.
Natural Antibiotic Resistance of Bacteria Isolated from Larvae of the Oil Fly,
Helaeomyia petrolei
Authors: Kadavy Dana R. , Hornby Jacob M. , Haverkost Terry , Nickerson Kenneth W.
Published: July 2002

Helaeomyia petrolei
(oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as
Providencia
spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as
Providencia rettgeri
exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine
Providencia
strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (
Morganella morganii
) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10
8
cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on
Pseudomonas aeruginosa
,
Escherichia coli
,
Staphylococcus aureus
, or
Candida albicans
cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 μg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene–50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.
Application of Siderotyping for Characterization of
Pseudomonas tolaasii
and “
Pseudomonas reactans
” Isolates Associated with Brown Blotch Disease of Cultivated Mushrooms
Authors: Munsch Patricia , Geoffroy Valerie A. , Alatossava Tapani , Meyer Jean-Marie
Published: July 2002

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of
P. tolaasii
and “
P. reactans
.” The bacteria, isolated from cultivated
Agaricus bisporus
or
Pleurotus ostreatus
mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401–407, 1979) and their pathogenicity towards
A. bisporus
and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen
P. tolaasii
isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 “
P. reactans
” isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some “
P. reactans
” isolates, with reference strains belonging to the
P. fluorescens
biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one
P. tolaasii
siderovar and one “
P. reactans
” siderovar were found.
Occurrence of Antimicrobial Resistance in Fish-Pathogenic and Environmental Bacteria Associated with Four Danish Rainbow Trout Farms
Authors: Schmidt Anja S. , Bruun Morten S. , Dalsgaard Inger , Pedersen Karl , Larsen Jens L.
Published: July 2002

Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark. Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88
Flavobacterium psychrophilum
isolates and 134
Yersinia ruckeri
isolates) and 313 motile
Aeromonas
isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples. MICs were obtained applying a standardized agar dilution method. A markedly decreased susceptibility of
F. psychrophilum
isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected
Y. ruckeri
isolates remained largely sensitive to all therapeutic substances. Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads. High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.
Phylogenetic Analysis of and Oligonucleotide Probe Development for Eikelboom Type 021N Filamentous Bacteria Isolated from Bulking Activated Sludge
Authors: Kanagawa Takahiro , Kamagata Yoichi , Aruga Shinobu , Kohno Tetsuro , Horn Matthias , Wagner Michael
Published: July 2002

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized
Thiothrix
species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of
Proteobacteria
. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-
Thiothrix
cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.
Simultaneous Detection of
Salmonella
Strains and
Escherichia coli
O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture
Authors: Sharma Vijay K. , Carlson Steve A.
Published: July 2002

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic
Salmonella
strains and
Escherichia coli
O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes
sipB
and
sipC
of
Salmonella
and an intragenic segment of gene
eae
of
E. coli
O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of
Salmonella enterica
serovar Typhimurium or
E. coli
O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in ≤4 h after enrichment.
Estimation of the Yield Coefficient of
Pseudomonas
sp. Strain DP-4 with a Low Substrate (2,4-Dichlorophenol [DCP]) Concentration in a Mineral Medium from Which Uncharacterized Organic Compounds Were Eliminated by a Non-DCP-Degrading Organism
Authors: Tarao Mitsunori , Seto Masayuki
Published: July 2002

The yield coefficient (YC) of
Pseudomonas
sp. strain DP-4, a 2,4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 μg of DCP-C ml
−1
. A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 μg of C ml of DCP
−1
and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 μg of C ml
−1
was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 μg of C ml
−1
was not degraded in mixed culture even by a high density, 10
5
CFU ml
−1
, of DP-4. When glucose was added to this mixed culture to a final concentration of 1 μg of C ml
−1
, the initial concentration of 0.01 μg of C ml of DCP
−1
was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 μg of C ml
−1
. The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 μg of C ml of DCP
−1
was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.
Bacteriocin Production with
Lactobacillus amylovorus
DCE 471 Is Improved and Stabilized by Fed-Batch Fermentation
Authors: Callewaert Raf , De Vuyst Luc
Published: July 2002

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by
Lactobacillus amylovorus
DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.
Selected Bacterial Strains Protect
Artemia
spp. from the Pathogenic Effects of
Vibrio proteolyticus
CW8T2
Authors: Verschuere Laurent , Heang Hanglamong , Criel Godelieve , Sorgeloos Patrick , Verstraete Willy
Published: July 2002

In this study
Vibrio proteolyticus
CW8T2 has been identified as a virulent pathogen for
Artemia
spp. Its infection route has been visualized with transmission electron microscopy. The pathogen affected microvilli and gut epithelial cells, disrupted epithelial cell junctions, and reached the body cavity, where it devastated cells and tissues. In vivo antagonism tests showed that preemptive colonization of the culture water with nine selected bacterial strains protected
Artemia
juveniles against the pathogenic effects. Two categories of the selected strains could be distinguished: (i) strains providing total protection, as no mortality occurred 2 days after the experimental infection with
V. proteolyticus
CW8T2, with strain LVS8 as a representative, and (ii) strains providing partial protection, as significant but not total mortality was observed, with strain LVS2 as a representative. The growth of
V. proteolyticus
CW8T2 in the culture medium was slowed down in the presence of strains LVS2 and LVS8, but growth suppression was distinctly higher with LVS8 than with LVS2. It was striking that the strains that gave only partial protection against the pathogen in the in vivo antagonism test showed also a restricted capability to colonize the
Artemia
compared to the strains providing total protection. The in vivo antagonism tests and the filtrate experiments showed that probably no extracellular bacterial compounds were involved in the protective action but that the living cells were required to protect
Artemia
against
V. proteolyticus
CW8T2.
A New Shiga Toxin 2 Variant (Stx2f) from
Escherichia coli
Isolated from Pigeons
Authors: Schmidt Herbert , Scheef Jürgen , Morabito Stefano , Caprioli Alfredo , Wieler Lothar H. , Karch Helge
Published: July 2002

We have isolated Shiga toxin (Stx)-producing
Escherichia coli
(STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated

stx
2f

. This gene is most similar to
sltIIva
of patient
E. coli
O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the

stx
2f

gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to
E. coli
pathogenic to humans may occur.
Interspecific Variability in Sensitivity to UV Radiation and Subsequent Recovery in Selected Isolates of Marine Bacteria
Authors: Arrieta Jesús María , Weinbauer Markus G. , Herndl Gerhard J.
Published: July 2002

The interspecific variability in the sensitivity of marine bacterial isolates to UV-B (295- to 320-nm) radiation and their ability to recover from previous UV-B stress were examined. Isolates originating from different microenvironments of the northern Adriatic Sea were transferred to aged seawater and exposed to artificial UV-B radiation for 4 h and subsequently to different radiation regimens excluding UV-B to determine the recovery from UV-B stress. Bacterial activity was assessed by thymidine and leucine incorporation measurements prior to and immediately after the exposure to UV-B and after the subsequent exposure to the different radiation regimens. Large interspecific differences among the 11 bacterial isolates were found in the sensitivity to UV-B, ranging from 21 to 92% inhibition of leucine incorporation compared to the bacterial activity measured in dark controls and from 14 to 84% for thymidine incorporation. Interspecific differences in the recovery from the UV stress were also large. An inverse relation was detectable between the ability to recover under dark conditions and the recovery under photosynthetic active radiation (400 to 700 nm). The observed large interspecific differences in the sensitivity to UV-B radiation and even more so in the subsequent recovery from UV-B stress are not related to the prevailing radiation conditions of the microhabitats from which the bacterial isolates originate. Based on our investigations on the 11 marine isolates, we conclude that there are large interspecific differences in the sensitivity to UV-B radiation and even larger differences in the mechanisms of recovery from previous UV stress. This might lead to UV-mediated shifts in the bacterioplankton community composition in marine surface waters.
A Binding Site for
Bacillus thuringiensis
Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests
Authors: Rausell Carolina , Martínez-Ramírez Amparo Consuelo , García-Robles Inmaculada , Real María Dolores
Published: July 2002

The insecticidal activity and receptor binding properties of
Bacillus thuringiensis
Cry1A toxins towards the forest pests
Thaumetopoea pityocampa
(processionary moth) and
Lymantria monacha
(nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar
T. pityocampa
larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with
125
I-labelled Cry1Ab and brush border membrane vesicles from
T. pityocampa
first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in
L. monacha
showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of
T. pityocampa
and
L. monacha
, correlating in
T. pityocampa
with a decrease in Cry1Ab toxicity with increasing age.
Anaerobic Naphthalene Degradation by Microbial Pure Cultures under Nitrate-Reducing Conditions
Authors: Rockne Karl J. , Chee-Sanford Joanne C. , Sanford Robert A. , Hedlund Brian P. , Staley James T. , Strand Stuart E.
Published: July 2002

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added
14
C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the
14
C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to
Pseudomonas stutzeri
and that NAP-4 was closely related to
Vibrio pelagius
. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.
Involvement of Two Plasmids in Fenitrothion Degradation by
Burkholderia
sp. Strain NF100
Authors: Hayatsu Masahito , Hirano Motoko , Tokuda Shinichi
Published: July 2002

A bacterium capable of utilizing fenitrothion (
O
,
O
-dimethyl
O
-4-nitro-
m
-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus
Burkholderia
and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.
Metabolic Analysis of
Escherichia coli
in the Presence and Absence of the Carboxylating Enzymes Phosphoenolpyruvate Carboxylase and Pyruvate Carboxylase
Authors: Gokarn R. R. , Eiteman M. A. , Altman E.
Published: July 2002

Fermentation patterns of
Escherichia coli
with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the
Rhizobium etli pyc
gene in
E. coli
(JCL1242/pTrc99A-
pyc
) restored the succinate producing ability of
E. coli ppc
null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-
pyc
than by cells which overproduced PPC (JCL1242/pPC201,
ppc
+
), even though PPC activity in cell extracts of JCL1242/pPC201 (
ppc
+
) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-
pyc
. Flux calculations indicate that during anaerobic metabolism the
pyc
+
strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the
ppc
+
strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.
Toluene Monooxygenase-Catalyzed Epoxidation of Alkenes
Authors: McClay Kevin , Fox Brian G. , Steffan Robert J.
Published: July 2002

Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of
Escherichia coli
expressing the cloned toluene-4-monooxygenase (T4MO) of
Pseudomonas mendocina
KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of
P. mendocina
KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 0.33 μmol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.
Cryptosporidiumspp. in Domestic Dogs: the “Dog” Genotype
Authors: Morgan Una M. , Xiao Lihua , Monis Paul , Fall Abbie , Irwin Peter J. , Fayer Ronald , Denholm Karen M. , Limor Josef , Lal Altaf , Thompson R. C. Andrew
Published: July 2002

Genetic and phylogenetic characterization ofCryptosporidiumisolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derivedCryptosporidiumisolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the “dog” genotype is, in fact, a valid species.
Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine
Authors: Gevertz Diane , Telang Anita J. , Voordouw Gerrit , Jenneman Gary E.
Published: July 2002

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O
2
). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO
2
as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO
2
. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division
Proteobacteria
, with CVO most closely related to
Thiomicrospira denitrifcans
and FWKO B most closely related to members of the genus
Arcobacter
. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.
Detection of Astroviruses, Enteroviruses, and Adenovirus Types 40 and 41 in Surface Waters Collected and Evaluated by the Information Collection Rule and an Integrated Cell Culture-Nested PCR Procedure
Authors: Chapron Christopher D. , Ballester Nicola A. , Fontaine Justin H. , Frades Christine N. , Margolin Aaron B.
Published: July 2002

We evaluated the use of an integrated cell culture-reverse transcription-PCR (ICC-RT-PCR) procedure coupled with nested PCR to detect human astroviruses, enteroviruses, and adenovirus types 40 and 41 in surface water samples that were collected and evaluated by using the Information Collection Rule (ICR) method. The results obtained with the ICC-RT-PCR–nested PCR method were compared to the results obtained with the total culturable virus assay–most-probable-number (TCVA-MPN) method, the method recommended by the U.S. Environmental Protection Agency for monitoring viruses in surface and finished waters. Twenty-nine ICR surface water samples were analyzed. Viruses were concentrated by using filter adsorption-beef extract elution and organic flocculation techniques, and then the preparations were evaluated for viruses by visualizing cytopathic effects in the Buffalo green monkey kidney (BGMK) cell line. In the ICC-RT-PCR–nested PCR technique we used Caco-2 cells to propagate astroviruses and enteroviruses (ICC step), and we used BGMK cells to propagate adenovirus types 40 and 41, as well as enteroviruses. Fifteen of the 29 samples (51.7%) were positive for astrovirus as determined by the ICC-RT-PCR–nested PCR method, and eight of these samples (27.5%) contained infectious astrovirus. Seventeen of the 29 samples (58.6%) were positive for enteroviruses when the BGMK cell line was used, and six (27.6%) of these samples were determined to be infectious. Fourteen of the 29 samples (48.3%) were positive for adenovirus types 40 and 41, and 11 (37.9%) of these samples were determined to be infectious. Twenty-seven of the 29 samples (93.1%) were positive for a virus, and 19 (68.9%) of the samples were positive for an infectious virus. Only 5 of the 29 samples (17.2%) were positive as determined by the TCVA-MPN method. The ICC-RT-PCR–nested PCR method provided increased sensitivity compared to the TCVA-MPN method.
Molecular Cloning, Characterization, and Differential Expression of a Glucoamylase Gene from the Basidiomycetous Fungus
Lentinula edodes
Authors: Zhao J. , Chen Y. H. , Kwan H. S.
Published: July 2002

The complete nucleotide sequence of putative glucoamylase gene
gla1
from the basidiomycetous fungus
Lentinula edodes
strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The
gla1
gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the
gla1
gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in
L. edodes
strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for
gla1
gene expression shows that expression of
gla1
was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus.
Phylogenetic Diversity among Geographically Dispersed
Chlamydiales
Endosymbionts Recovered from Clinical and Environmental Isolates of
Acanthamoeba
spp
Authors: Fritsche Thomas R. , Horn Matthias , Wagner Michael , Herwig Russell P. , Schleifer Karl-Heinz , Gautom Romesh K.
Published: July 2002

The recently proposed reorganization of the order
Chlamydiales
and description of new taxa are broadening our perception of this once narrowly defined taxon. We have recovered four strains of gram-negative cocci endosymbiotic in
Acanthamoeba
spp., representing 5% of the
Acanthamoeba
sp. isolates examined, which displayed developmental life cycles typical of members of the
Chlamydiales
. One of these endosymbiont strains was found stably infecting an amoebic isolate recovered from a case of amoebic keratitis in North America, with three others found in acanthamoebae recovered from environmental sources in North America (two isolates) and Europe (one isolate). Analyses of nearly full-length 16S rRNA gene sequences of these isolates by neighbor joining, parsimony, and distance matrix methods revealed their clustering with other members of the
Chlamydiales
but in a lineage separate from those of the genera
Chlamydia
,
Chlamydophila
,
Simkania
, and
Waddlia
(sequence similarities, <88%) and including the recently described species
Parachlamydia acanthamoebae
(sequence similarities, 91.2 to 93.1%). With sequence similarities to each other of 91.4 to 99.4%, these four isolates of intra-amoebal endosymbionts may represent three distinct species and, perhaps, new genera within the recently proposed family
Parachlamydiaceae
. Fluorescently labeled oligonucleotide probes targeted to 16S rRNA signature regions were able to readily differentiate two groups of intra-amoebal endosymbionts which corresponded to two phylogenetic lineages. These results reveal significant phylogenetic diversity occurring among the
Chlamydiales
in nontraditional host species and supports the existence of a large environmental reservoir of related species. Considering that all described species of
Chlamydiales
are known to be pathogenic, further investigation of intra-amoebal parachlamydiae as disease-producing agents is warranted.
Isolation of Tannin-Degrading Lactobacilli from Humans and Fermented Foods
Authors: Osawa Ro , Kuroiso Keiko , Goto Satoshi , Shimizu Akira
Published: July 2002

Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to
Lactobacillus plantarum
,
L. paraplantarum
, and
L. pentosus
. Additional studies on a range of
Lactobacillus
species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.
Physiological and Regulatory Effects of Controlled Overproduction of Five Cold Shock Proteins of
Lactococcus lactis
MG1363
Authors: Wouters Jeroen A. , Mailhes Marielle , Rombouts Frank M. , de Vos Willem M. , Kuipers Oscar P. , Abee Tjakko
Published: July 2002

The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of
Lactococcus lactis
were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total protein) was obtained. Northern blot analysis revealed low abundance of the
cspC
transcript, indicating that the stability of
cspC
mRNA is low. The limited overproduction of CspA is likely to be caused by low stability of CspA since when there was an Arg-Pro mutation at position 58, the level of CspA production increased. Using two-dimensional gel electrophoresis, it was found that upon overproduction of the CSPs several proteins, including a number of cold-induced proteins of
L. lactis
, were induced. Strikingly, upon overproduction of CspC induction of CspB, putative CspF, and putative CspG was also observed. Overproduction of CspB and overproduction of CspE result in increased survival when
L. lactis
is frozen (maximum increases, 10- and 5-fold, respectively, after 4 freeze-thaw cycles). It is concluded that in
L. lactis
CSPs play a regulatory role in the cascade of events that are initiated by cold shock treatment and that they either have a direct protective effect during freezing (e.g., RNA stabilization) or induce other factors involved in the freeze-adaptive response or both.
Polyhydroxyalkanoate Degradation Is Associated with Nucleotide Accumulation and Enhances Stress Resistance and Survival ofPseudomonas oleovoransin Natural Water Microcosms
Authors: Ruiz Jimena A. , López Nancy I. , Fernández Rubén O. , Méndez Beatriz S.
Published: July 2002

Pseudomonas oleovoransGPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248–277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of thephaZmutation restored the wild-type phenotype.
Clonal Populations of Thermotolerant
Enterobacteriaceae
in Recreational Water and Their Potential Interference with Fecal
Escherichia coli
Counts
Authors: McLellan Sandra L. , Daniels Annette D. , Salmore Alissa K.
Published: July 2002

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for
Escherichia coli
enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of β-glucuronidase activity revealed that these clonal isolates were
Klebsiella
,
Citrobacter
, and
Enterobacter
spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be
E. coli
, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. β-Glucuronidase activity was critical in distinguishing
E. coli
from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that
E. coli
is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.