bluuebunny/crossref_metadata_2025 huggingface dataset:
Classification of Antibiotic Resistance Patterns of Indicator Bacteria by Discriminant Analysis: Use in Predicting the Source of Fecal Contamination in Subtropical Waters
Authors: Harwood Valerie J. , Whitlock John , Withington Victoria
Published: July 2002

The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida.
Virulence Genes in Environmental Strains of
Vibrio cholerae
Authors: Chakraborty Soumen , Mukhopadhyay Asish K. , Bhadra Rupak Kumar , Ghosh Amar Nath , Mitra Rupak , Shimada Toshio , Yamasaki Shinji , Faruque Shah M. , Takeda Yoshifumi , Colwell Rita R. , Nair G. Balakrish
Published: July 2002

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The
ctxAB
and
tcpA
genes are known to play a cardinal role in maintaining virulence in
Vibrio cholerae
, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including
ctxAB
and
tcpA
, as well as
toxR
and
toxT
, which are involved in the regulation of virulence, in environmental strains of
V. cholerae
cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental
V. cholerae
strains. Sequencing of a part of the
tcpA
gene carried by an environmental strain showed 97.7% homology to the
tcpA
gene of the classical biotype of
V. cholerae
O1. Strains carrying the
tcpA
gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying
ctxAB
genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of
V. cholerae
, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of
V. cholerae
.
Leaky
Lactococcus
Cultures That Externalize Enzymes and Antigens Independently of Culture Lysis and Secretion and Export Pathways
Authors: Walker Shirley A. , Klaenhammer Todd R.
Published: July 2002

A novel system that leaks β-galactosidase (β-gal) without a requirement for secretion or export signals was developed in
Lactococcus lactis
by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage φ31 is an 888-bp fragment (P
15A10
) encoding the transcriptional activator. When a high-copy-number P
15A10
::
lacZ.st
fusion was introduced into
L. lactis
strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant β-gal activity were detected in the supernatant (approximately 85% of the total β-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P
15A10
fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of β-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage φ31 late promoter, into the host genome of MG1363 yielded a similar “leaky” phenotype, indicating that holin and lysin might play a critical role in the release of β-gal into the medium. In addition to β-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.
Occurrence of Tn
4371
-Related Mobile Elements and Sequences in (Chloro)biphenyl-Degrading Bacteria
Authors: Springael Dirk , Ryngaert Annemie , Merlin Christophe , Toussaint Ariane , Mergeay Max
Published: July 2002

Tn
4371
, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in
Ralstonia eutropha
A5, displays a modular structure including a phage-like integrase gene (
int
), a
Pseudomonas
-like (chloro)biphenyl catabolic gene cluster (
bph
), and RP4- and Ti-plasmid-like transfer genes (
trb
) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40–54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn
4371
in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn
4371
-related sequences were never detected on endogenous plasmids. Although the gene probes containing only
bph
sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all β-proteobacteria, displayed hybridization patterns similar to the Tn
4371 bph
cluster. Homology between Tn
4371
and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn
4371
. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn
4371
containing the
int
gene. The
bph
catabolic determinant of the two strains displaying homology to the Tn
4371
transfer genes and a third strain isolated from the A5 area could be mobilized to a
R. eutropha
recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn
4371
homologous sequences previously identified in their genome. Our observations show that the
bph
genes present on Tn
4371
are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the β-proteobacteria. On the other hand, Tn
4371
-related mobile elements carrying
bph
genes are apparently only found in isolates from the environment that provided the Tn
4371
-bearing isolate A5.
Antimicrobial Properties of Garlic Oil against Human Enteric Bacteria: Evaluation of Methodologies and Comparisons with Garlic Oil Sulfides and Garlic Powder
Authors: Ross Z. M. , O'Gara E. A. , Hill D. J. , Sleightholme H. V. , Maslin D. J.
Published: July 2002

The antimicrobial effects of aqueous garlic extracts are well established but those of garlic oil (GO) are little known. Methodologies for estimating the antimicrobial activity of GO were assessed and GO, GO sulfide constituents, and garlic powder (GP) were compared in tests against human enteric bacteria. Test methodologies were identified as capable of producing underestimates of GO activity. Antimicrobial activity was greater in media lacking tryptone or cysteine, suggesting that, as for allicin, GO effects may involve sulfhydryl reactivity. All bacteria tested, which included both gram-negative and -positive bacteria and pathogenic forms, were susceptible to garlic materials. On a weight-of-product basis, 24 h MICs for GO (0.02 to 5.5 mg/ml, 62 enteric isolates) and dimethyl trisulfide (0.02 to 0.31 mg/ml, 6 enteric isolates) were lower than those for a mixture of diallyl sulfides (0.63 to 25 mg/ml, 6 enteric isolates) and for GP, which also exhibited a smaller MIC range (6.25 to 12.5 mg/ml, 29 enteric isolates). Viability time studies of GO and GP against
Enterobacter aerogenes
showed time- and dose-dependent effects. Based upon its thiosulfinate content, GP was more active than GO against most bacteria, although some properties of GO are identified as offering greater therapeutic potential. Further exploration of the potential of GP and GO in enteric disease control appears warranted.
Response of the Endophytic Diazotroph
Gluconacetobacter diazotrophicus
on Solid Media to Changes in Atmospheric Partial O
2
Pressure
Authors: Pan Bo , Vessey J. Kevin
Published: July 2002

Gluconacetobacter diazotrophicus
is an N
2
-fixing endophyte isolated from sugarcane.
G. diazotrophicus
was grown on solid medium at atmospheric partial O
2
pressures (pO
2
) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO
2
(5 to 60 kPa). Nitrogenase activity was measured by H
2
evolution in N
2
-O
2
and in Ar-O
2
, and respiration rate was measured by CO
2
evolution in N
2
-O
2
. To validate the use of H
2
production as an assay for nitrogenase activity, a non-N
2
-fixing (Nif
−
) mutant of
G. diazotrophicus
was tested and found to have a low rate of uptake hydrogenase (Hup
+
) activity (0.016 ±  0.009 μmol of H
2
10
10
cells
−1
h
−1
) when incubated in an atmosphere enriched in H
2
. However, Hup
+
activity was not detectable under the normal assay conditions used in our experiments.
G. diazotrophicus
fixed nitrogen at all atmospheric pO
2
tested. However, when the assay atmospheric pO
2
was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO
2
for nitrogenase activity was 0 to 20 kPa above the pO
2
at which the bacteria had been grown. As atmospheric pO
2
was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO
2
was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO
2
from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O
2
, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O
2
protection mechanism of nitrogenase activity. Our study demonstrates that colonies of
G. diazotrophicus
can fix N
2
at a wide range of atmospheric pO
2
and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO
2
.
In Situ Characterization of
Nitrospira
-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants
Authors: Daims Holger , Nielsen Jeppe L. , Nielsen Per H. , Schleifer Karl-Heinz , Wagner Michael
Published: July 2002

Uncultivated
Nitrospira
-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of
Nitrospira
-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the
Nitrospira
phylum including all publicly available sequences was constructed. This analysis revealed that the genus
Nitrospira
consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus
Nitrospira
, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of
Nitrospira
-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the
Nitrospira
microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by
Nitrospira
-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the
Nitrospira
-like bacteria in bioreactor samples took up inorganic carbon (as HCO
3
−
or as CO
2
) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the
Nitrospira
-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.
Biodegradation of Methyltert-Butyl Ether by a Pure Bacterial Culture
Authors: Hatzinger Paul B. , McClay Kevin , Vainberg Simon , Tugusheva Marina , Condee Charles W. , Steffan Robert J.
Published: July 2002

Biodegradation of methyltert-butyl ether (MTBE) by the hydrogen-oxidizing bacteriumHydrogenophaga flavaENV735 was evaluated. ENV735 grew slowly on MTBE ortert-butyl alcohol (TBA) as sole sources of carbon and energy, but growth on these substrates was greatly enhanced by the addition of a small amount of yeast extract. The addition of H2did not enhance or diminish MTBE degradation by the strain, and MTBE was only poorly degraded or not degraded by type strains ofHydrogenophagaor hydrogen-oxidizing enrichment cultures, respectively. MTBE degradation activity was constitutively expressed in ENV735 and was not greatly affected by formaldehyde, carbon monoxide, allyl thiourea, or acetylene. MTBE degradation was inhibited by 1-amino benzotriazole and butadiene monoepoxide. TBA degradation was inducible by TBA and was inhibited by formaldehyde at concentrations of >0.24 mM and by acetylene but not by the other inhibitors tested. These results demonstrate that separate, independently regulated genes encode MTBE and TBA metabolism in ENV735.
Shared Binding Sites in Lepidoptera for
Bacillus thuringiensis
Cry1Ja and Cry1A Toxins
Authors: Herrero Salvador , González-Cabrera Joel , Tabashnik Bruce E. , Ferré Juan
Published: July 2002

Bacillus thuringiensis
toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to
B. thuringiensis
toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (
Plutella xylostella
) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family:
Cacyreus marshalli
(Lycaenidae),
Lobesia botrana
(Tortricidae),
Manduca sexta
(Sphingidae),
Pectinophora gossypiella
(Gelechiidae),
P. xylostella
(Plutellidae), and
Spodoptera exigua
(Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in
Heliothis virescens
(Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.
Nickel-Resistance-Based Minitransposons: New Tools for Genetic Manipulation of Environmental Bacteria
Authors: Taghavi Safieh , Delanghe Hilde , Lodewyckx Cindy , Mergeay Max , van der Lelie Daniël
Published: July 2002

The
ncc
and
nre
nickel resistance determinants from
Ralstonia eutropha
-like strain 31A were used to construct mini-Tn
5
transposons. Broad host expression of nickel resistance was observed for the
nre
minitransposons in members of the α, β, and γ subclasses of the
Proteobacteria
, while the
ncc
minitransposons expressed nickel resistance only in
R. eutropha
-like strains.
Impact of UV Radiation on Bacterioplankton Community Composition
Authors: Winter Christian , Moeseneder Markus M. , Herndl Gerhard J.
Published: July 2002

The potential effect of UV radiation on the composition of coastal marine bacterioplankton communities was investigated. Dilution cultures with seawater collected from the surface mixed layer of the coastal North Sea were exposed to different ranges of natural or artificial solar radiation for up to two diurnal cycles. The composition of the bacterioplankton community prior to exposure was compared to that after exposure to the different radiation regimes using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA and 16S ribosomal DNA. Only minor changes in the composition of the bacterial community in the different radiation regimes were detectable. Sequencing of selected bands obtained by DGGE revealed that some species of the
Flexibacter-Cytophaga-Bacteroides
(FCB) group were sensitive to UV radiation while other species of the FCB group were resistant. Overall, only up to ≈10% of the operational taxonomic units present in the dilution cultures appeared to be affected by UV radiation. Thus, we conclude that UV radiation has little effect on the composition of coastal marine bacterioplankton communities in the North Sea.
Mobilization of Selenite by
Ralstonia metallidurans
CH34
Authors: Roux Murielle , Sarret Géraldine , Pignot-Paintrand Isabelle , Fontecave Marc , Coves Jacques
Published: July 2002

Ralstonia metallidurans
CH34 (formerly
Alcaligenes eutrophus
CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals.
R. metallidurans
CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of
R. metallidurans
CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.
Spatial Arrangements and Associative Behavior of Species in an In Vitro Oral Biofilm Model
Authors: Guggenheim M. , Shapiro S. , Gmür R. , Guggenheim B.
Published: July 2002

The spatial arrangements and associative behavior of
Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus
, and
Streptococcus oralis
strains in an in vitro model of supragingival plaque were determined. Using species-specific fluorescence-labeled antibodies in conjunction with confocal laser scanning microscopy, the volumes and distribution of the five strains were assessed during biofilm formation. The volume-derived cell numbers of each strain correlated well with respective culture data. Between 15 min and 64 h, populations of each strain increased in a manner reminiscent of batch growth. The microcolony morphologies of all members of the consortium and their distributions within the biofilm were characterized, as were interspecies associations. Biofilms formed 15 min after inoculation consisted principally of single nonaggregated cells. All five strains adhered strongly to the saliva-conditioned substratum, and therefore, coadhesion played no role during the initial phase of biofilm formation. This observation does not reflect the results of in vitro coaggregation of the five strains, which depended upon the nature of the suspension medium. While the possibility cannot be excluded that some interspecies associations observed at later stages of biofilm formation were initiated by coadhesion, increase in bacterial numbers appeared to be largely a growth phenomenon regulated by the prevailing cultivation conditions.
Cold-Adapted β-Galactosidase from the Antarctic Psychrophile
Pseudoalteromonas haloplanktis
Authors: Hoyoux A. , Jennes I. , Dubois P. , Genicot S. , Dubail F. , François J. M. , Baise E. , Feller G. , Gerday C.
Published: July 2002

The β-galactosidase from the Antarctic gram-negative bacterium
Pseudoalteromonas haloplanktis
TAE 79 was purified to homogeneity. The nucleotide sequence and the NH
2
-terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated
M
r
of 118,068. This β-galactosidase shares structural properties with
Escherichia coli
β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters.
P. haloplanktis
β-galactosidase was expressed in
E. coli
, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both
P. haloplanktis
wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that
P. haloplanktis
β-galactosidase can outperform the current commercial β-galactosidase from
Kluyveromyces marxianus
var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.
Molecular Screening of
Enterococcus
Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates
Authors: Eaton Tracy J. , Gasson Michael J.
Published: July 2002

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus
Enterococcus
is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of
Enterococcus faecalis
,
E. faecium
, and
E. durans
. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the
E. faecalis
and
E. faecium
strains. Medical
E. faecalis
strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the
E. faecalis
strains tested possessed multiple determinants (between 6 and 11).
E. faecium
strains were generally free of virulence determinants, with notable exceptions. Significantly,
esp
and
gelE
determinants were identified in
E. faecium
medical strains. These virulence determinants have not previously been identified in
E. faecium
strains and may result from regional differences or the evolution of pathogenic
E. faecium
. Phenotypic testing revealed the existence of apparently silent
gelE
and
cyl
genes. In
E. faecalis
, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of
Enterococcus
spp. in foods requires careful safety evaluation.
Enterocin P Selectively Dissipates the Membrane Potential of
Enterococcus faecium
T136
Authors: Herranz C. , Chen Y. , Chung H.-J. , Cintas L. M. , Hernández P. E. , Montville T. J. , Chikindas M. L.
Published: July 2002

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against
Listeria monocytogenes
. The bacteriocin was purified from the culture supernatant of
Enterococcus faecium
P13, and its molecular mechanism of action against the sensitive strain
E. faecium
T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from
E. faecium
T136-derived liposomes.
Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores
Authors: Williams Roger H. , Ward Elaine , McCartney H. Alastair
Published: July 2002

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, usingPenicillium roquefortias a model fungus, are described.P. roquefortispores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. ThreeP. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection ofP. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities ofP. roquefortispores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced.P. roquefortiDNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10P. roquefortispores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.
Development of Aflatoxin B
1
-Lysine Adduct Monoclonal Antibody for Human Exposure Studies
Authors: Wang Jia-Sheng , Abubaker Salahaddin , He Xia , Sun Guiju , Strickland Paul T. , Groopman John D.
Published: July 2002

Mouse monoclonal antibodies were developed against a synthetic aflatoxin B
1
(AFB)-lysine–cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(λ). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(
N
7
-guanyl)-9-hydroxy-AFB > aflatoxin M
1
> aflatoxin Q
1
. IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when
3
H-AFB–lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(
N
7
-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and
3
H-AFB–lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer.
Diversity and Characterization of Sulfate-Reducing Bacteria in Groundwater at a Uranium Mill Tailings Site
Authors: Chang Yun-Juan , Peacock Aaron D. , Long Philip E. , Stephen John R. , McKinley James P. , Macnaughton Sarah J. , Hussain A. K. M. Anwar , Saxton Arnold M. , White David C.
Published: July 2002

Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from δ-Proteobacteria, gram-positive organisms, and the
Nitrospira
division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0.
Desulfotomaculum
and
Desulfotomaculum
-like sequences were the most dominant DSR genes detected. Those belonging to SRB within δ-Proteobacteria were mainly recovered from low-uranium (≤302 ppb) samples. One
Desulfotomaculum
-like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (
P
= 0.0001). This strong association indicates that
Desulfotomaculum
has remarkable tolerance and adaptation to high levels of uranium and suggests the organism's possible involvement in natural attenuation of uranium. The in situ activity level of
Desulfotomaculum
in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.
Rapid Approach To Determine
rrn
Arrangement in
Salmonella
Serovars
Authors: Helm R. Allen , Maloy Stanley
Published: July 2002

A PCR method was developed by which to rapidly and accurately determine the
rrn
arrangement of
Salmonella enterica
serovars. Primers were designed to the genomic regions flanking each of the seven
rrn
operons
.
PCR analysis using combinations of these primers will distinguish each of the possible arrangements of the
rrn
skeleton.
Construction and Environmental Release of a
Sinorhizobium meliloti
Strain Genetically Modified To Be More Competitive for Alfalfa Nodulation
Authors: van Dillewijn Pieter , Soto Marı́a José , Villadas Pablo J. , Toro Nicolás
Published: July 2002

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a
Sinorhizobium meliloti
PutA
−
mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of
S. meliloti
on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492–498, 1995). In this work, we investigated whether the
putA
gene could be used as a means of increasing the competitiveness of
S. meliloti
strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the
putA
gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this
putA
gene construct increased the competitiveness of
S. meliloti
in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of
S. meliloti
strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified
putA
gene for samples that were analyzed after 34 days but not for samples that were analyzed later.
nifH
Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs
Authors: Auman Ann J. , Speake Catherine C. , Lidstrom Mary E.
Published: July 2002

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N
2
as a nitrogen source. However, no sequences are available for
nif
genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains.
nifH
gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the
nifH
sequences from the type I and type II strains cluster with
nifH
sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated
nifH
sequences from three
Methylomonas
strains show high identity (95 to 99%) to several published translated environmental
nifH
sequences PCR amplified from rice roots and a freshwater lake. The translated
nifH
sequences from the type II strains show high identity (94 to 99%) to published translated
nifH
sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.
Survival of Clinical and Poultry-Derived Isolates of
Campylobacter jejuni
at a Low Temperature (4°C)
Authors: Chan Kam Fai , Le Tran Huyen , Kanenaka R. Y. , Kathariou S.
Published: July 2002

Campylobacter jejuni
is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31°C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of
C. jejuni
(10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4°C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at −20°C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4°C) tolerance.
Production of Recombinant α-Galactosidases in
Thermus thermophilus
Authors: Fridjonsson Olafur , Mattes Ralf
Published: July 2002

A
Thermus thermophilus
selector strain for production of thermostable and thermoactive α-galactosidase was constructed. For this purpose, the native α-galactosidase gene (
agaT
) of
T. thermophilus
TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of
agaT
by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (α-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal
+
phenotype was assumed to be essential for growth on melibiose. In a Gal
−
background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant α-galactosidase. Moreover, the AgaT
−
strain had to be Km
s
for establishment of the plasmids containing α-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT
−
Gal
+
Km
s
) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous α-galactosidase in
T. thermophilus
, the isogenes
agaA
and
agaB
of
Bacillus stearothermophilus
KVE36 were cloned into an
Escherichia coli-Thermus
shuttle vector. The region containing the
E. coli
plasmid sequence (pUC-derived vector) was deleted before transformation of
T. thermophilus
with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.
Phylogeny of Culturable Estuarine Bacteria Catabolizing Riverine Organic Matter in the Northern Baltic Sea
Authors: Kisand Veljo , Cuadros Rocio , Wikner Johan
Published: July 2002

The objective of our study was to isolate and determine the phylogenetic affiliation of culturable estuarine bacteria capable of catabolizing riverine dissolved organic matter (RDOM) under laboratory conditions. Additions of RDOM consistently promoted the growth of estuarine bacteria in carbon-limited dilution cultures, with seasonal variation in growth rates and yields. At least 42 different taxa were culturable on solid agar media and, according to quantitative DNA-DNA hybridizations, constituted 32 to 89% of the total bacterial number in the enriched treatments. Five species in the
Cytophaga-Flexibacter-Bacteroides
group and one in the γ-proteobacteria phylogenetic group (
Marinomonas
sp.) were numerically dominant during the stationary phase of the RDOM-enriched dilution cultures but not in the control cultures. Four of the isolates in
Cytophaga-Flexibacter-Bacteroides
group were putatively affiliated with the genus
Flavobacterium
. All dominating isolates were determined to be new species based on comparison to the current databases. The same group of species dominated independently of the season investigated, suggesting a low diversity of bacteria catabolizing RDOM in the estuary. It also suggested a broad tolerance of the dominating species to seasonal variation in hydrography, chemistry, and competition with other species. Taken together, our results suggest that a limited group of bacteria, mainly in the
Flavobacterium
genus, played an important role in introducing new energy and carbon to the marine system in the northern Baltic Sea.
Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes
Authors: Gutiérrez Ana , del Río José C. , Martínez-Íñigo María J. , Martínez María J. , Martínez Ángel T.
Published: July 2002

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with
Ceriporiopsis subvermispora
(up to 24 mg per 100 g of wood) but also were produced by
Phlebia radiata
,
Pleurotus pulmonarius
, and
Bjerkandera adusta
. We hypothesize that three main alkylitaconic acids (tetradecylitaconic,
cis
-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.
Molecular Biological Detection and Characterization of
Clostridium
Populations in Municipal Landfill Sites
Authors: Van Dyke M. I. , McCarthy A. J.
Published: July 2002

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the
Clostridium thermocellum
and
C. leptum
groups. In contrast, the
C. botulinum
group was never detected, and the
C. coccoides
-
C. lentocellum
group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.
Ochratoxin Production by the
Aspergillus ochraceus
Group and
Aspergillus alliaceus
Authors: Bayman Paul , Baker James L. , Doster Mark A. , Michailides Themis J. , Mahoney Noreen E.
Published: July 2002

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the
Aspergillus ochraceus
group (section
Circumdati
) and
Aspergillus alliaceus
from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards,
A. ochraceus
and
Aspergillus melleus
were the most common species. No field isolates of
A. ochraceus
or
A. melleus
produced ochratoxin A above the level of detection (0.01 μg/ml). All
A. alliaceus
isolates produced ochratoxin A, up to 30 μg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with
A. alliaceus
contained ochratoxin A, figs infected with the
A. ochraceus
group had little or none, and figs infected with
Penicillium
had none. These results suggest that the little-known species
A. alliaceus
is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.
Involvement of
Rhodocyclus
-Related Organisms in Phosphorus Removal in Full-Scale Wastewater Treatment Plants
Authors: Zilles Julie L. , Peccia Jordan , Kim Myeong-Woon , Hung Chun-Hsiung , Noguera Daniel R.
Published: July 2002

The participation of organisms related to
Rhodocyclus
in full-scale enhanced biological phosphorus removal (EBPR) was investigated. By using fluorescent in situ hybridization techniques, the communities of
Rhodocyclus
-related organisms in two full-scale wastewater treatment plants were estimated to represent between 13 and 18% of the total bacterial population. However, the fractions of these communities that participated in polyphosphate accumulation depended on the type of treatment process evaluated. In a University of Cape Town EBPR process, the percentage of
Rhodocyclus
-related cells that contained polyphosphate was about 20% of the total bacterial population, but these cells represented as much as 73% of the polyphosphate-accumulating organisms (PAOs). In an aerated-anoxic EBPR process,
Rhodocyclus
-related PAOs were less numerous, accounting for 6% of the total bacterial population and 26% of the total PAO population. In addition, 16S ribosomal DNA sequences 99.9% similar to the sequences of
Rhodocyclus
-related organisms enriched in acetate-fed bench-scale EBPR reactors were recovered from both full-scale plants. These results confirmed the involvement of
Rhodocyclus
-related organisms in EBPR and demonstrated their importance in full-scale processes. In addition, the results revealed a significant correlation between the type of EBPR process and the PAO community.
Conservation of the 17-Kilodalton Antigen Gene within the Genus
Bartonella
Authors: Sweger Debra , Resto-Ruiz Sandra , Johnson David P. , Schmiederer Michael , Hawke Noel , Anderson Burt
Published: July 2002

The 17-kDa antigen of
Bartonella henselae
has previously been shown to elicit a strong humoral immune response in patients with cat scratch disease (CSD) and to be useful in screening human serum samples for CSD. In this study, PCR amplification of genes homologous to the 17-kDa antigen gene of
B. henselae
was performed using genomic DNAs from several species of
Bartonella
, including the currently recognized human pathogens. Amplicons of similar size were demonstrated using the following chromosomal DNA templates:
B. henselae
(two strains),
B. quintana
(two strains),
B. elizabethae
,
B. clarridgeiae
,
B. vinsonii
subsp.
vinsonii
, and
B. vinsonii
subsp.
berkhoffii
. No evidence of a
B. bacilliformis
homolog of the 17-kDa antigen gene was obtained using multiple primer pairs. DNA sequencing revealed open reading frames capable of coding for proteins with sizes similar to that of the 17-kDa antigen of
B. henselae
in all of the amplicons; however, extensive sequence divergence across the genus was noted. Cloning of the amplified products into pUC19 resulted in recombinants that directed synthesis of homologs of the 17-kDa protein. Immunoblot analysis using human sera from CSD cases demonstrated very little cross-reactivity among different species for this protein. In contrast, immunoblots using rabbit serum raised to the recombinant
B. henselae
antigen showed extensive cross-reactivity with the proteins of other
Bartonella
species. The data suggest that the use of the 17-kDa antigen as a serologic reagent may allow the development of more specific diagnostic assays. Furthermore, the nucleotide sequences from the various versions of the 17-kDa antigen gene should be useful for rapid identification of
Bartonella
at the species level.