bluuebunny/crossref_metadata_2025_split huggingface dataset:
PCR Assay of the
groEL
Gene for Detection and Differentiation of
Bacillus cereus
Group Cells
Authors: Chang Yu-Hsiu , Shangkuan Yung-Hui , Lin Hung-Chi , Liu Hwan-Wun 
Published: August 2003

ABSTRACT

Strains of species in the
Bacillus cereus
group are potentially enterotoxic. Thus, the detection of all
B. cereus
group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the
B. cereus
group, we explored the potential of the
groEL
gene as a phylogenetic marker. A phylogenetic analysis of the
groEL
sequences of 78
B. cereus
group strains revealed that the
B. cereus
group strains were split into two major clusters, one including six
B. mycoides
and one
B. pseudomycoides
(cluster II) and the other including two
B. mycoides
and the rest of the
B. cereus
group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The
sodA
gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the
sodA
sequences showed substantial similarity to the tree constructed from the
groEL
sequences. Based on the
groEL
sequences, a PCR assay for detection and identification of
B. cereus
group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the
B. cereus
group strains. RFLP with
Mbo
I was identical for all the
B. cereus
group strains analyzed, while RFLP with
Mfe
I or
Pst
I classified all
B. cereus
and
B. thuringiensis
strains into two groups. All cluster II
B. mycoides
and
B. pseudomycoides
strains could be discriminated from other
B. cereus
group bacteria by restriction analysis with
Tsp
RI.
Isolation and Characterization of
Campylobacter
Bacteriophages from Retail Poultry
Authors: Atterbury Robert J. , Connerton Phillippa L. , Dodd Christine E. R. , Rees Catherine E. D. , Connerton Ian F. 
Published: August 2003

ABSTRACT

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of
Campylobacter
in poultry production. To this end, we have developed a procedure to recover
Campylobacter
bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized
Campylobacter
phage (i.e., NCTC 12674). By using this method, we have shown that
Campylobacter
phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous
Campylobacter
phages. Thirty-four
Campylobacter
bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (
P
< 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that
Campylobacter
bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.
Biocontrol of
Listeria monocytogenes
on Fresh-Cut Produce by Treatment with Lytic Bacteriophages and a Bacteriocin
Authors: Leverentz Britta , Conway William S. , Camp Mary J. , Janisiewicz Wojciech J. , Abuladze Tamuna , Yang Ming , Saftner Robert , Sulakvelidze Alexander 
Published: August 2003

ABSTRACT

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied
Listeria monocytogenes
populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic,
L. monocytogenes
-specific phages via two phage application methods, spraying and pipetting, on
L. monocytogenes
populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced
L. monocytogenes
populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced
L. monocytogenes
populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced
L. monocytogenes
populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of
L. monocytogenes
.
Osmoregulation in the Parasitic Protozoan
Tritrichomonas foetus
Authors: Maroulis Sarah L. , Schofield Philip J. , Edwards Michael R. 
Published: August 2003

ABSTRACT

Tritrichomonas foetus
was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of
T. foetus
intracellular amino acids revealed a pool size (65 mM) that was similar to that of
Trichomonas vaginalis
but was considerably smaller than those of
Giardia intestinalis
and
Crithidia luciliae
. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the
T. foetus
RVI. The
T. foetus
intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K
+
concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the
T. foetus
RVI was enhanced by elevated external K
+
concentrations and was inhibited when K
+
and/or Cl
−
was absent from the medium. The results suggested that the well-documented Na
+
-K
+
-2Cl
−
cotransport system was responsible for the K
+
influx activated during the RVI. However, inhibitors of Na
+
-K
+
-2Cl
−
cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K
+
influx in
T. foetus
.
Identification and Characterization of Phenylpyruvate Decarboxylase Genes in
Saccharomyces cerevisiae
Authors: Vuralhan Zeynep , Morais Marcos A. , Tai Siew-Leng , Piper Matthew D. W. , Pronk Jack T. 
Published: August 2003

ABSTRACT

Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding α-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in
Saccharomyces cerevisiae
, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway.
S. cerevisiae
possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity:
YDR380w/ARO10
,
YDL080C/THI3
,
PDC1
,
PDC5
, and
PDC6
. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (
ARO10
) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that
ARO10
was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene
THI3
did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (
PDC1
,
PDC5
, and
PDC6
) for the alternative activity. The

K
m

and
V
max
values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering of the Ehrlich pathway.
Effects of Cultivation Conditions on Folate Production by Lactic Acid Bacteria
Authors: Sybesma Wilbert , Starrenburg Marjo , Tijsseling Linda , Hoefnagel Marcel H. N. , Hugenholtz Jeroen 
Published: August 2003

ABSTRACT

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly.
Lactococcus lactis
,
Streptococcus thermophilus
, and
Leuconostoc
spp. all produced folate, while most
Lactobacillus
spp., with the exception of
Lactobacillus plantarum
, were not able to produce folate. Folate production was further investigated in
L
.
lactis
as a model organism for metabolic engineering and in
S
.
thermophilus
for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production.
S
.
thermophilus
excreted more folate than
L
.
lactis
, presumably as a result of differences in the number of glutamyl residues of the folate produced. In
S
.
thermophilus
5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in
L
.
lactis
5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in
S
.
thermophilus
, but pH had no effect on folate excretion by
L
.
lactis
. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of
p
-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.
Ubiquity and Persistence of
Escherichia coli
in a Midwestern Coastal Stream
Authors: Byappanahalli Muruleedhara , Fowler Melanie , Shively Dawn , Whitman Richard 
Published: August 2003

ABSTRACT

Dunes Creek, a small Lake Michigan coastal stream that drains sandy aquifers and wetlands of Indiana Dunes, has chronically elevated
Escherichia coli
levels along the bathing beach near its outfall. This study sought to understand the sources of
E. coli
in Dunes Creek's central branch. A systematic survey of random and fixed sampling points of water and sediment was conducted over 3 years.
E. coli
concentrations in Dunes Creek and beach water were significantly correlated. Weekly monitoring at 14 stations during 1999 and 2000 indicated chronic loading of
E. coli
throughout the stream. Significant correlations between
E. coli
numbers in stream water and stream sediment, submerged sediment and margin, and margin and 1 m from shore were found. Median
E. coli
counts were highest in stream sediments, followed by bank sediments, sediments along spring margins, stream water, and isolated pools; in forest soils,
E. coli
counts were more variable and relatively lower. Sediment moisture was significantly correlated with
E. coli
counts. Direct fecal input inadequately explains the widespread and consistent occurrence of
E. coli
in the Dunes Creek watershed; long-term survival or multiplication or both seem likely. The authors conclude that (i)
E. coli
is ubiquitous and persistent throughout the Dunes Creek basin, (ii)
E. coli
occurrence and distribution in riparian sediments help account for the continuous loading of the bacteria in Dunes Creek, and (iii) ditching of the stream, increased drainage, and subsequent loss of wetlands may account for the chronically high
E. coli
levels observed.
Real-Time PCR for Simultaneous Detection and Quantification of
Borrelia burgdorferi
in Field-Collected
Ixodes scapularis
Ticks from the Northeastern United States
Authors: Wang Guiqing , Liveris Dionysios , Brei Brandon , Wu Hongyan , Falco Richard C. , Fish Durland , Schwartz Ira 
Published: August 2003

ABSTRACT

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the
Borrelia burgdorferi
-specific
recA
gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete,
B
.
burgdorferi
, in field-collected
Ixodes scapularis
ticks. The sensitivity of qPCR for detection of
B
.
burgdorferi
DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498
I
.
scapularis
ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (
P
= 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of
B
.
burgdorferi
(
P
= 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of
k
= 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of
B
.
burgdorferi
infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
Rapid Identification ofCandidaSpecies by Using Nuclear Magnetic Resonance Spectroscopy and a Statistical Classification Strategy
Authors: Himmelreich Uwe , Somorjai Ray L. , Dolenko Brion , Lee Ok Cha , Daniel Heide-Marie , Murray Ronan , Mountford Carolyn E. , Sorrell Tania C. 
Published: August 2003

ABSTRACTNuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genusCandidato characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates ofCandida albicans,C. glabrata,C. krusei,C. parapsilosis, andC. tropicalisresulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates ofC. parapsilosisandC. glabratawith unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.
Engineering
Deinococcus geothermalis
for Bioremediation of High-Temperature Radioactive Waste Environments
Authors: Brim Hassan , Venkateswaran Amudhan , Kostandarithes Heather M. , Fredrickson James K. , Daly Michael J. 
Published: August 2003

ABSTRACT

Deinococcus geothermalis
is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile
Deinococcus radiodurans
, which is being engineered for in situ bioremediation of radioactive wastes. We report that
D
.
geothermalis
is transformable with plasmids designed for
D
.
radiodurans
and have generated a Hg(II)-resistant
D
.
geothermalis
strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally,
D
.
geothermalis
is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55°C.
Microbial Quality and Direct PCR Identification of Lactic Acid Bacteria and Nonpathogenic Staphylococci from Artisanal Low-Acid Sausages
Authors: Aymerich T. , Martín B. , Garriga M. , Hugas M. 
Published: August 2003

ABSTRACTDetection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment,Lactobacillus sakeiandLactobacillus curvatuswere detected in 11.8% of the samples, andLactobacillus plantarumandStaphylococcus xylosuswere detected in 17.6%. Enriched samples allowed the detection ofL. sakeiandS. xylosusin all of the samples (100%) and ofEnterococcus faeciumin 11.8% of the sausages. The percentages ofL. curvatus,L. plantarum,Staphylococcus carnosus, andStaphylococcus epidermidisvaried depending on the sausage type.L. curvatuswas detected in 80% of fuets and in 57% of chorizos.L. plantarumwas found in 50% of fuets and 100% of chorizos.S. epidermidiswas detected in only 11.8% of fuets, andS. carnosuswas detected in only 5.9% of chorizos.Lactococcus lactis,Staphylococcus warneri, andStaphylococcus simulanswere not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts ofEnterobacteriaceaeand did not contain any of the food-borne pathogens assayed.
A Newly Discovered
Bacteroides
Conjugative Transposon, CTnGERM1, Contains Genes Also Found in Gram-Positive Bacteria
Authors: Wang Yanping , Wang Gui-Rong , Shelby Aikiesha , Shoemaker Nadja B. , Salyers Abigail A. 
Published: August 2003

ABSTRACT

Results of a recent study of antibiotic resistance genes in human colonic
Bacteroides
strains suggested that gene transfer events between members of this genus are fairly common. The identification of
Bacteroides
isolates that carried an erythromycin resistance gene,
ermG
, whose DNA sequence was 99% identical to that of an
ermG
gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into
Bacteroides
species from other genera. Six of seven
ermG-
containing
Bacteroides
strains tested were able to transfer
ermG
by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying
ermG
in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in
Bacteroides
isolates—CTns of the CTnERL/CTnDOT family—which sometimes carry another type of
erm
gene,
ermF
. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the
ermG
-containing strains but also to DNA from strains that did not carry
ermG
. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired
ermG
. The percentage of G+C content of the
ermG
region was significantly lower than that of the chromosome of
Bacteroides
species—an indication that CTnGERM1 may have entered
Bacteroides
strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered
Bacteroides
species relatively recently. One of the genes located upstream of e
rmG
encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from
Streptococcus pyogenes
. Our having found >90% sequence identity of two upstream genes, including
mefA
, and the remnants of two transposon-carried genes downstream of
ermG
with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.
Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding ofCryptosporidium parvumin Adult Beef Cattle
Authors: Atwill E. R. , Hoar B. , das Graças Cabral Pereira M. , Tate K. W. , Rulofson F. , Nader G. 
Published: August 2003

ABSTRACTOur primary goal was to generate an accurate estimate of the daily environmental loading rate ofCryptosporidium parvumoocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding ofC.parvumoocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces−1, with a 54% probability of detecting just one oocyst g of feces−1. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive forC.parvumwas 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces−1. The estimated environmental loading rate ofC.parvumranged from 3,900 to 9,200 oocysts cow−1day−1, which is substantially less than a previous estimate of 1.7 × 105oocysts cow−1day−1(range of 7.7 × 104to 2.3 × 105oocysts cow−1day−1) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol.2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.