bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Resistance to Ciprofloxacin, Rifampin, and Mupirocin in Methicillin-Susceptible and -Resistant
Staphylococcus aureus
Isolates
Authors: Schmitz Franz-Josef , Fluit Ad C. , Hafner Dieter , Beeck Andreas , Perdikouli Mirella , Boos Mechthild , Scheuring Sybille , Verhoef Jan , Köhrer Karl , Von Eiff Christof
Published: July 2002

A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described for
Staphylococcus aureus
. Differences in resistance rates may be explainable by a higher spontaneous mutation rate (MR) or a faster development of resistance (DIFF) in methicillin-resistant
S. aureus
(MRSA). No differences in MR, DIFF, and mutations in
grlA
and
gyrA
were detected between methicillin-susceptible
S. aureus
and MRSA. The higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of different mutations and may be the consequence of clonal spread of multiresistant MRSA.
Variation within the
vat
(E) Allele of
Enterococcus faecium
Isolates from Retail Poultry Samples
Authors: Simjee S. , McDermott P. F. , Wagner D. D. , White D. G.
Published: July 2002

In a survey of retail meat samples, twelve quinupristin-dalfopristin-resistant (MICs, ≥4 mg/liter)
Enterococcus faecium
isolates that carried a
vat
(E) gene were recovered. DNA sequence comparison revealed five new variations in the
vat
(E) allele among 12 isolates, which were designated
vat
(E-4) through
vat
(E-8); two isolates had
vat
(E-1). There was no correlation between the number of base changes and the quinupristin-dalfopristin MIC.
Activities of New Macrolides and Fluoroquinolones against
Mycobacterium ulcerans
Infection in Mice
Authors: Bentoucha Abdelhalim , Robert Jérôme , Dega Hervé , Lounis Nacer , Jarlier Vincent , Grosset Jacques
Published: July 2002

Mice infected in the left hind footpad with 5 log
10
acid-fast bacilli of
Mycobacterium ulcerans
were divided into an untreated control group and 17 treatment groups that received one of the following regimens for 4 weeks (all doses in milligrams per kilogram): 100 mg of azithromycin (AZM), 100 mg of clarithromycin (CLR), or 50 mg of AZM for a duration of 5 days a week (daily), three times a week, or once weekly. In addition, the following regimens were administered daily: 100 mg of telithromycin (TLM), sparfloxacin (SPX), or moxifloxacin (MOX); 200 mg of levofloxacin (LVX); 100 mg of streptomycin (STR) or amikacin (AMK); 10 mg of rifampin (RIF); and the combination of 10 mg of RIF and 100 mg of AMK (RIF+AMK). After completion of treatment, mice were observed for 30 weeks. The effectiveness of treatment regimens was assessed in terms of the delay in median time to footpad swelling in treated mice compared with that in the untreated controls. Clear-cut bactericidal activity, i.e., an observed delay in footpad swelling that exceeded the period of treatment, was observed in the STR-, AMK-, and RIF+AMK-treated mice. However, all mice treated with either AMK or STR alone had swollen footpads before the end of the 30-week observation period, suggesting regrowth of
M. ulcerans
. In contrast, 50% of the mice treated with the RIF+AMK combination exhibited no lesion even after 30 weeks, suggesting cure. The remaining regimens could be assigned to one of three groups: (i) no activity (50 mg of AZM, 100 mg of AZM thrice weekly, TLM, and LVX); (ii) bacteriostatic activity, i.e., a delay in footpad swelling shorter than the 4-week treatment duration (100 mg of AZM daily or once weekly, CLR thrice or once weekly, and MOX); or (iii) weak bactericidal activity (CLR daily and SPX). The RIF+AMK combination and possibly RIF+STR warrant further study for the treatment
of M. ulcerans
infection in humans.
Susceptibilities of Oral Bacteria and Yeast to Mammalian Cathelicidins
Authors: Guthmiller Janet M. , Vargas Kaaren G. , Srikantha Rupasree , Schomberg Lori L. , Weistroffer Paula L. , McCray Paul B. , Tack Brian F.
Published: July 2002

The effects of cathelicidins against oral bacteria and clinically important oral yeasts are not known. We tested the susceptibilities of
Actinobacillus actinomycetemcomitans
,
Fusobacterium nucleatum
,
Porphyromonas gingivalis
,
Streptococcus sanguis
,
Candida krusei
,
Candida tropicalis
and
Candida albicans
to the following cathelicidins: FALL39, SMAP29, and CAP18. SMAP29 and CAP18 were antimicrobial, whereas FALL39 did not exhibit antimicrobial activity. Future studies are needed to determine the potential use of these antimicrobial peptides in prevention and treatment of oral infections.
Quantitative PCR Assay To Measure
Aspergillus fumigatus
Burden in a Murine Model of Disseminated Aspergillosis: Demonstration of Efficacy of Caspofungin Acetate
Authors: Bowman J. C. , Abruzzo G. K. , Anderson J. W. , Flattery A. M. , Gill C. J. , Pikounis V. B. , Schmatz D. M. , Liberator P. A. , Douglas C. M.
Published: July 2002

Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-β-
d
-glucan, an essential component of the cell wall of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-β-
d
-glucan synthesis inhibitors against
Aspergillus fumigatus
has been evaluated in animal models of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy.
A. fumigatus
added to naı̈ve, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of
A. fumigatus
was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log
10
units greater than mean values determined by CFU measurement. When used to monitor disease progression in infected mice, the qPCR assay detected an increase of nearly 4 log
10
conidial equivalents/g of kidney between days 1 and 4 following infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with
Candida albicans
, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated aspergillosis with either caspofungin or amphotericin B reduced the
A. fumigatus
burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against
A. fumigatus
.
Bactericidal Activities of Two Daptomycin Regimens against Clinical Strains of Glycopeptide Intermediate-Resistant
Staphylococcus aureus
, Vancomycin-Resistant
Enterococcus faecium
, and Methicillin-Resistant
Staphylococcus aureus
Isolates in an In Vitro Pharmacodynamic Model with Simulated Endocardial Vegetations
Authors: Akins Ronda L. , Rybak Michael J.
Published: July 2002

Daptomycin is an investigational lipopeptide antibiotic active against gram-positive organisms. The mechanism of action is unique, resulting in interference with cell membrane transport. The bactericidal activity of daptomycin was evaluated against glycopeptide-intermediate susceptible
Staphylococcus aureus
(GISA), vancomycin-resistant
Enterococcus faecium
(VREF), and methicillin-resistant
S. aureus
(MRSA) in an in vitro infection model with simulated endocardial vegetations. Simulated regimens of daptomycin at 6 mg/kg/day (D6) and 10 mg/kg/day (D10) were utilized. MICs and MBCs for daptomycin were determined in the absence and in the presence of albumin with the following results (MIC/MBC): for GISA-992, 0.5/1.0 and 16/16; for VREF-590, 2.0/2.0 and 32/32; and for MRSA-494, 0.25/0.25 and 1.0/4.0 μg/ml, respectively. During the first 8 h daptomycin significantly reduced the inoculum for all organisms. Daptomycin at 6 mg/kg/day and 10 mg/kg/day had log
10
CFU/g reductions of 5 and 6, 3.4 and 5, and 6.4 and 6.5 by 8 h for GISA-992, VREF-590, and MRSA-494, respectively. Against both GISA-992 and VREF-590, the D10 regimen achieved the limit of detection at 72 h, with D6 regimens showing slight regrowth. A concentration-dependent killing effect was noted to occur, with daptomycin demonstrating a more rapid and greater kill from the D10 versus the D6 regimen. The results of this study suggest that daptomycin demonstrates significant (
P
< 0.05) activity against gram-positive organisms in a simulated sequestered infection site.
Synergy of Histone-Derived Peptides of Coho Salmon with Lysozyme and Flounder Pleurocidin
Authors: Patrzykat Aleksander , Zhang Lijuan , Mendoza Valentina , Iwama George K. , Hancock Robert E. W.
Published: July 2002

Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activity, can be induced in coho salmon (
Oncorhynchus kisutch
) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against
Vibrio anguillarum
and
Aeromonas salmonicida
, a four-step protein purification protocol was used to isolate and identify antibacterial fractions from bacterially challenged coho salmon mucus and blood. The purification consisted of extraction with hot acetic acid, extraction and concentration on a C
18
cartridge, gel filtration, and reverse-phase chromatography on a C
18
column. N-terminal amino acid sequence analyses revealed that both the blood and the mucus antimicrobial fractions demonstrated identity with the N terminus of trout H1 histone. Mass spectroscopic analysis indicated the presence of the entire histone, as well as fragments thereof, including a 26-amino-acid N-terminal segment. These fractions inhibited the growth of antibiotic-supersuscptible
Salmonella enterica
serovar Typhimurium, as well as
A. salmonicida
and
V. anguillarum
. Synthetic peptides identical to the N-terminally acetylated or C-terminally amidated 26-amino-acid fragment were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. The peptides bound specifically to anionic lipid monolayers. However, synergy with pleurocidin did not appear to occur at the cell membrane level. The synergistic activities of inducible histone peptides indicate that they play an important role in the first line of salmon defenses against infectious pathogens and that while some histone fragments may have direct antimicrobial effects, others improve existing defenses.
Antimonial-Mediated DNA Fragmentation inLeishmania infantumAmastigotes
Authors: Sereno Denis , Holzmuller Philippe , Mangot Isabelle , Cuny Gérard , Ouaissi Ali , Lemesre Jean-Loup
Published: July 2002

The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes ofLeishmania infantumat low concentrations (10 μg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 μg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.
Echinococcus multilocularis
Alkaline Phosphatase as a Marker for Metacestode Damage Induced by In Vitro Drug Treatment with Albendazole Sulfoxide and Albendazole Sulfone
Authors: Stettler Marianne , Siles-Lucas Mar , Sarciron Elisabeth , Lawton Philippe , Gottstein Bruno , Hemphill Andrew
Published: July 2002

Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm
Echinococcus multilocularis
. The disease affects the human liver and occasionally other organs and is fatal if treatment is unsuccessful. The present chemotherapy of AE is based on the administration of benzimidazole carbamate derivatives, such as mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some cases, parasitostatic rather than parasiticidal, and the recurrence rate is rather high. Therefore, chemotherapy usually involves the lifelong uptake of massive doses of albendazole and new treatment options are urgently needed. In order to avoid costly and time-consuming animal experimentation, a first step in searching for novel parasiticidal compounds could be the in vitro drug screening of novel compounds by employing metacestode cultivation. However, presently used techniques (e.g., transmission electron microscopy) for determination of parasite viability involve costly equipment and time-consuming preparation of rather large amounts of parasite material. We therefore searched for a parasite marker which can be easily traced and the presence or absence of which is indicative of parasite viability. In this study we show that the increase of
E. multilocularis
alkaline phosphatase activity in culture supernatants during in vitro drug treatment with albendazole derivatives correlates with the progressive degeneration and destruction of the metacestode tissue. The inexpensive and rapid assay presented here will serve as an ideal tool for performing first-round in vitro tests on the efficacy of a large number of antiparasitic compounds.
Risk Factors for Emergence of Resistance to Broad-Spectrum Cephalosporins among
Enterobacter
spp
Authors: Kaye Keith S. , Cosgrove Sara , Harris Anthony , Eliopoulos George M. , Carmeli Yehuda
Published: July 2002

Among 477 patients with susceptible
Enterobacter
spp., 49 subsequently harbored third-generation cephalosporin-resistant
Enterobacter
spp. Broad-spectrum cephalosporins were independent risk factors for resistance (relative risk [OR] = 2.3,
P
= 0.01); quinolone therapy was protective (OR = 0.4,
P
= 0.03). There were trends toward decreased risk for resistance among patients receiving broad-spectrum cephalosporins and either aminoglycosides or imipenem. Of the patients receiving broad-spectrum cephalosporins, 19% developed resistance.
Bacilysocin, a Novel Phospholipid Antibiotic Produced by
Bacillus subtilis
168
Authors: Tamehiro Norimasa , Okamoto-Hosoya Yoshiko , Okamoto Susumu , Ubukata Makoto , Hamada Masa , Naganawa Hiroshi , Ochi Kozo
Published: July 2002

We have found a novel phospholipid antibiotic (named bacilysocin) which accumulates within (or associates with) the cells of
Bacillus subtilis
168 and determined the structure by nuclear magnetic resonance and mass spectrometry analyses. The structure of bacilysocin elucidated was 1-(12-methyltetradecanoyl)-3-phosphoglyceroglycerol. Bacilysocin demonstrated antimicrobial activity, especially against certain fungi. Production of bacilysocin commenced immediately after growth ceased and before the formation of heat-resistant spores. The production of bacilysocin was completely blocked when the
ytpA
gene, which encodes a protein homologous to lysophospholipase, was disrupted, but blockage of the
ytpA
gene did not significantly affect growth. Sporulation was also impaired, with a 10-fold reduction in heat-resistant spore titers being detected. Since the
ytpA
disruptant actually lacked phospholipase activity, we propose that the YtpA protein functions as an enzyme for the biosynthesis of bacilysocin.
Gemifloxacin Is Efficacious against Penicillin-Resistant and Quinolone-Resistant Pneumococci in Experimental Meningitis
Authors: Cottagnoud Philippe , Acosta Fernando , Cottagnoud Marianne , Täuber Martin G.
Published: July 2002

In experimental rabbit meningitis, gemifloxacin penetrated inflamed meninges well (22 to 33%) and produced excellent bactericidal activity (change in log
10
[Δlog
10
] CFU/ml/h, −0.68 ± 0.30 [mean and standard deviation]), even superior to that of the standard regimen of ceftriaxone plus vancomycin (−0.49 ± 0.09 Δlog
10
CFU/ml/h), in the treatment of meningitis due to a penicillin-resistant pneumococcal strain (MIC, 4 mg/liter). Even against a penicillin- and quinolone-resistant strain, gemifloxacin showed good bactericidal activity (−0.48 ± 0.16 Δlog
10
CFU/ml/h). The excellent antibacterial activity of gemifloxacin was also confirmed by time-kill assays over 8 h in vitro.
Cytochrome
c
3
Mutants of
Desulfovibrio desulfuricans
Authors: Rapp-Giles Barbara J. , Casalot Laurence , English R. Samuel , Ringbauer Joseph A. , Dolla Alain , Wall Judy D.
Published: July 2002

To explore the physiological role of tetraheme cytochrome
c
3
in the sulfate-reducing bacterium
Desulfovibrio desulfuricans
G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome
c
3
in the electron transport pathway from pyruvate to sulfate or sulfite in
D. desulfuricans
G20.
Flow Cytometry for Determination of the Efficacy of Contact Lens Disinfecting Solutions against
Acanthamoeba
spp
Authors: Borazjani Roya N. , May Lauren L. , Noble Judith A. , Avery Simon V. , Ahearn Donald G.
Published: July 2002

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of
Acanthamoeba castellanii
and
Acanthamoeba polyphaga
. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10
5
to 10
6
/ml) at 22°C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.
Bacterial Reductive Dissolution of Crystalline Fe(III) Oxide in Continuous-Flow Column Reactors
Authors: Roden Eric E. , Urrutia Matilde M. , Mann Carroll J.
Published: July 2002

Bacterial reductive dissolution of synthetic crystalline Fe(III) oxide-coated sand was studied in continuous-flow column reactors in comparison with parallel batch cultures. The cumulative amount of aqueous Fe(II) exported from the columns over a 6-month incubation period corresponded to (95.0 ± 3.7)% (n = 3) of their original Fe(III) content. Wet-chemical analysis revealed that only (6.5 ± 3.2)% of the initial Fe(III) content remained in the columns at the end of the experiment. The near-quantitative removal of Fe was visibly evidenced by extensive bleaching of color from the sand in the columns. In contrast to the column reactors, Fe(II) production quickly reached an asymptote in batch cultures, and only (13.0 ± 2.2)% (n = 3) of the Fe(III) oxide content was reduced. Sustained bacterial-cell growth occurred in the column reactors, leading to the production and export of a quantity of cells 100-fold greater than that added during inoculation. Indirect estimates of cell growth, based on the quantity of Fe(III) reduced, suggest that only an approximate doubling of initial cell abundance was likely to have occurred in the batch cultures. Our results indicate that removal of biogenic Fe(II) via aqueous-phase transport in the column reactors decreased the passivating influence of surface-bound Fe(II) on oxide reduction activity, thereby allowing a dramatic increase in the extent of Fe(III) oxide reduction and associated bacterial growth. These findings have important implications for understanding the fate of organic and inorganic contaminants whose geochemical behavior is linked to Fe(III) oxide reduction.
Prokaryotic Diversity in
Zostera noltii
-Colonized Marine Sediments
Authors: Cifuentes Ana , Antón Josefa , Benlloch Susana , Donnelly Andrew , Herbert Rodney A. , Rodríguez-Valera Francisco
Published: July 2002

The diversity of microorganisms present in a sediment colonized by the phanerogam
Zostera noltii
has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for
Bacteria
and
Archaea
. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain
Bacteria
were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was δ-
Proteobacteria
related to sulfate-reducing bacteria. The second most abundant group (27%) was γ-
Proteobacteria
, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of
Crenarchaeota
and
Euryarchaeota
, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the
Bacteria
organisms. None of these sequences was closely related to cultured
Archaea
organisms.
Viral Density and Virus-to-Bacterium Ratio in Deep-Sea Sediments of the Eastern Mediterranean
Authors: Danovaro Roberto , Serresi Michela
Published: July 2002

Viruses are now recognized as a key component in pelagic systems, but their role in marine sediment has yet to be assessed. In this study bacterial and viral densities were determined at nine deep-sea stations selected from three main sites (i.e., the Sporades Basin, the Cretan Sea, and the Ierapetra Trench at depths of 1,232, 1,840, and 4,235 m, respectively) of the Eastern Mediterranean. The three areas were characterized by different phytopigment and biopolymeric carbon concentrations and by changes in the protein and carbohydrate pools. A gradient of increasing trophic conditions was observed from the Sporades Basin (North Aegean) to the Ierapetra Trench (South Aegean). Viral densities (ranging from 1 × 10
9
to 2 × 10
9
viruses ml of sediment
−1
) were significantly correlated to bacterial densities (
n
= 9,
r
2
= 0.647) and reached values up to 3 orders of magnitude higher than those generally reported for the water column. However, the virus-to-bacterium density ratio in deep-sea sediments was about 1 order of magnitude lower (range of 2 to 5, with a modal value of 2.6) than in pelagic environments. Virus density decreased vertically with depth in sediment cores at all stations and was below detection limits at the 10-cm depth of the abyssal sediments of the Ierapetra Trench. Virus density in the sediment apparently reflected a gradient of particle fluxes and trophic conditions, displaying the highest values in the Sporades Basin. The low virus-to-bacterium ratios and their inverse relationship with station depth suggest that the role played by viruses in controlling deep-sea benthic bacterial assemblages and biogeochemical cycles is less relevant than in pelagic systems.
Horizontal Heterogeneity of Denitrifying Bacterial Communities in Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis
Authors: Scala David J. , Kerkhof Lee J.
Published: July 2002

Although it is widely believed that horizontal patchiness exists in microbial sediment communities, determining the extent of variability or the particular members of the bacterial community which account for the observed differences among sites at various scales has not been routinely demonstrated. In this study, horizontal heterogeneity was examined in time and space for denitrifying bacteria in continental shelf sediments off Tuckerton, N.J., at the Rutgers University Long-Term Ecosystem Observatory (LEO-15). Characterization of the denitrifying community was done using PCR amplification of the nitrous oxide reductase (
nosZ
) gene combined with terminal restriction fragment length polymorphism analysis. Spatial scales from centimeters to kilometers were examined, while temporal variation was assayed over the course of 1995 to 1996. Sorenson's indices (pairwise similarity values) were calculated to permit comparison between samples. The similarities of benthic denitrifiers ranged from 0.80 to 0.85 for centimeter scale comparisons, from 0.52 to 0.79 for meter level comparisons, and from 0.23 to 0.53 for kilometer scale comparisons. Sorenson's indices for temporal comparisons varied from 0.12 to 0.74. A cluster analysis of the similarity values indicated that the composition of the denitrifier assemblages varied most significantly at the kilometer scale and between seasons at individual stations. Specific
nosZ
genes were identified which varied at centimeter, meter, or kilometer scales and may be associated with variability in meio- or macrofaunal abundance (centimeter scale), bottom topography (meter scale), or sediment characteristics (kilometer scale).
Parallel Processing of Substrate Correlates with Greater Functional Stability in Methanogenic Bioreactor Communities Perturbed by Glucose
Authors: Hashsham Syed A. , Fernandez Ana S. , Dollhopf Sherry L. , Dazzo Frank B. , Hickey Robert F. , Tiedje James M. , Criddle Craig S.
Published: July 2002

Parallel processing is more stable than serial processing in many areas that employ interconnected activities. This hypothesis was tested for microbial community function using two quadruplicate sets of methanogenic communities, each set having substantially different populations. The two communities were maintained at a mean cell residence time of 16 days and a mean glucose loading rate of 0.34 g/liter-day in variable-volume reactors. To test stability to perturbation, they were subjected to an instantaneous glucose pulse that resulted in a 6.8-g/liter reactor concentration. The pattern of accumulated products in response to the perturbation was analyzed for various measures of functional stability, including resistance, resilience, and reactivity for each product. A new stability parameter, “moment of amplification envelope,” was used to compare the soluble compound stability. These parameters indicated that the communities with predominantly parallel substrate processing were functionally more stable in response to the perturbation than the communities with predominantly serial substrate processing. The data also indicated that there was good replication of function under perturbed conditions; the degrees of replication were 0.79 and 0.83 for the two test communities.
Molecular Studies on the Ecology of
Listeria monocytogenes
in the Smoked Fish Processing Industry
Authors: Norton Dawn M. , McCamey Meghan A. , Gall Kenneth L. , Scarlett Janet M. , Boor Kathryn J. , Wiedmann Martin
Published: July 2002

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of
Listeria monocytogenes
in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for
L. monocytogenes
using a commercial PCR system (BAX for Screening/
Listeria monocytogenes
), including 57 (27.7%) environmental samples (
n
= 206), 8 (7.8%) raw material samples (
n
= 102), 23 (18.1%) samples from fish in various stages of processing(
n
= 127), and 7 (7.3%) finished product samples (
n
= 96).
L. monocytogenes
was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in
Listeria
Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of
L. monocytogenes
, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (
P
≤ 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different
L. monocytogenes
strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.
Percent G+C Profiling Accurately Reveals Diet-Related Differences in the Gastrointestinal Microbial Community of Broiler Chickens
Authors: Apajalahti Juha H. A. , Kettunen Anu , Bedford Michael R. , Holben William E.
Published: July 2002

Broiler chickens from eight commercial farms in Southern Finland were analyzed for the structure of their gastrointestinal microbial community by a nonselective DNA-based method, percent G+C-based profiling. The bacteriological impact of the feed source and in-farm whole-wheat amendment of the diet was assessed by percent G+C profiling. Also, a phylogenetic 16S rRNA gene (rDNA)-based study was carried out to aid in interpretation of the percent G+C profiles. This survey showed that most of the 16S rDNA sequences found could not be assigned to any previously known bacterial genus or they represented an unknown species of one of the taxonomically heterogeneous genera, such as
Ruminococcus
or
Clostridium
. The data from bacterial community profiling were analyzed by
t
-test, multiple linear regression, and principal-component statistical approaches. The percent G+C profiling method with appropriate statistical analyses detected microbial community differences smaller than 10% within each 5% increment of the percent G+C profiles. Diet turned out to be the strongest determinant of the cecal bacterial community structure. Both the source of feed and local feed amendment changed the bacteriological profile significantly, whereas profiles of individual farms with identical feed regimens hardly differed from each other. This suggests that the management of typical Finnish farms is relatively uniform or that hygiene on the farm, in fact, has little impact on the structure of the cecal bacterial community. Therefore, feed compounders should have a significant role in the modulation of gut microflora and consequently in prevention of gastrointestinal disorders in farm animals.
Biotransformations of Paralytic Shellfish Toxins by Bacteria Isolated from Bivalve Molluscs
Authors: Smith Elizabeth A. , Grant Faye , Ferguson Carolyn M. J. , Gallacher Susan
Published: July 2002

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.
Application of the 5′ Fluorogenic Exonuclease Assay (TaqMan) for Quantitative Ribosomal DNA and rRNA Analysis in Sediments
Authors: Stults Jennie R. , Snoeyenbos-West Oona , Methe Barbara , Lovley Derek R. , Chandler Darrell P.
Published: July 2002

In this study, we report on the development of quantitative PCR and reverse transcriptase PCR assays for the 16S rRNA of
Geobacter
spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. The lower detection limit of each assay was 5 to 50 fg of genomic DNA or ≤2 pg of 16S rRNA. TaqMan PCR spectral traces from uncontaminated, amended aquifer sediments were significantly lower (
P
< 0.0002) than traces for the external standard curve. We also observed a similar, significant decrease in mean quencher emissions for undiluted extracts relative to those for diluted extracts (
P
< 0.0001). If PCR enumerations were based solely upon the undiluted sample eluant, the TaqMan assay generated an inaccurate result even though the threshold cycle (
C

t

) measurements were precise and reproducible in the sediment extracts. Assay accuracy was significantly improved by employing a system of replicate dilutions and replicate analyses for both DNA and rRNA quantitation. Our results clearly demonstrate that fluorescence quenching and autofluorescence can significantly affect TaqMan PCR enumeration accuracy, with subsequent implications for the design and implementation of TaqMan PCR to sediments and related environmental samples.
Bile Salt Hydrolase Activity and Resistance to Toxicity of Conjugated Bile Salts Are Unrelated Properties in Lactobacilli
Authors: Moser Scott A. , Savage Dwayne C.
Published: July 2002

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous
Lactobacillus
spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.
Metabolic Engineering of the Morphology of
Aspergillus oryzae
by Altering Chitin Synthesis
Authors: Müller Christian , McIntyre Mhairi , Hansen Kim , Nielsen Jens
Published: July 2002

Morphology and α-amylase production during submerged cultivation were examined in a wild-type strain (A1560) and in strains of
Aspergillus oryzae
in which chitin synthase B (
chsB
) and chitin synthesis myosin A (
csmA
) have been disrupted (ChsB/G and CM101). In a flowthrough cell, the growth of submerged hyphal elements was studied online, making it possible to examine the growth kinetics of the three strains. The average tip extension rates of the CM101 and ChsB/G strains were 25 and 88% lower, respectively, than that of the wild type. The branching intensity in the CM101 strain was 25% lower than that in the wild type, whereas that in the ChsB/G strain was 188% higher. During batch cultivation, inseparable clumps were formed in the wild-type strain, while no or fewer large inseparable clumps existed in the cultivations of the ChsB/G and CM101 strains. The α-amylase productivity was not significantly different in the three strains. A strain in which the transcription of
chsB
could be controlled by the nitrogen source-regulated promoter
niiA
(NiiA1) was examined during chemostat cultivation, and it was found that the branching intensity could be regulated by regulating the promoter, signifying an important role for
chsB
in branching. However, the pattern of branching responded very slowly to the change in transcription, and increased branching did not affect α-amylase productivity. α-Amylase residing in the cell wall was stained by immunofluorescence, and the relationship between tip number and enzyme secretion is discussed.
Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics
Authors: Camilla Christophe , Mély Laurent , Magnan Antoine , Casano Brice , Prato Sabine , Debono Stephane , Montero Felix , Defoort Jean-Philippe , Martin Marie , Fert Vincent
Published: July 2002

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (
r
> 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (
P
= 0.003) and less IFN-γ (
P
= 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-γ than that of atopic nonasthmatic subjects (
P
= 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.
Dengue Virus Replicative Intermediate RNA Detection by Reverse Transcription-PCR
Authors: Vaughan Gilberto , Olivera Hiram , Santos-Argumedo Leopoldo , Landa Abraham , Briseño Baltasar , Escobar-Gutiérrez Alejandro
Published: July 2002

Dengue virus replication involves synthesis of a replicative intermediate RNA (RI-RNA), whose presence reveals an actual infection. We report on a simple and rapid reverse transcription-PCR for the detection of viral RI-RNA in infected cells. The product is demonstrated at 20 min postinfection. This method is useful for the study of virus-cell tropism.
Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity
Authors: Brodeur Bernard R. , Boyer Martine , Charlebois Isabelle , Hamel Josée , Couture France , Rioux Clément R. , Martin Denis
Published: July 2002

A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the
sip
gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6.84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the
sip
gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate.
Expression of the
Helicobacter pylori ureI
Gene Is Required for Acidic pH Activation of Cytoplasmic Urease
Authors: Scott David R. , Marcus Elizabeth A. , Weeks David L. , Lee Adrian , Melchers Klaus , Sachs George
Published: July 2002

ureI
encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of
Helicobacter pylori
and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced
H. pylori ureI
deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined
ureI
expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric
Helicobacter
species.
ureI
was found to be present in the genome of the gastric
Helicobacter
species and absent in the nongastric
Helicobacter
species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric
Helicobacter
species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric
Helicobacter
species. The
ureI
gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.
A Model of Infected Burn Wounds Using
Escherichia coli
O18:K1:H7 for the Study of Gram-Negative Bacteremia and Sepsis
Authors: Busch Nathan A. , Zanzot Emily M. , Loiselle Paul M. , Carter Edward A. , Allaire Jennifer E. , Yarmush Martin L. , Warren H. Shaw
Published: July 2002

A difficulty that has emerged in the development and preclinical evaluation of adjuvant therapies for gram-negative sepsis is the lack of easily studied animal models that closely mimic human infection. An objective of this study was to adapt a previously described model of infection in burned mice to rats with a defined bacterial strain of
Escherichia coli
. Challenge with two colonies of live
E. coli
O18:K1:H7 bacteria into an 8% full-thickness burn of the dorsal skin surface of rats produced predictable bacteremia at 24 to 48 h and 80 to 100% mortality at 3 to 4 days.
E. coli
O18:K1:H7 was approximately 10-million-fold more virulent than several other gram-negative bacterial strains. The model should be a useful tool in studying the pathogenicity of burn wound infections and in evaluating the efficacy of novel adjuvant therapies for gram-negative sepsis.
Coxiella burnetii
Exhibits Morphological Change and Delays Phagolysosomal Fusion after Internalization by J774A.1 Cells
Authors: Howe Dale , Mallavia Louis P.
Published: July 2002

Coxiella burnetii
, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in
C. burnetii
survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization of
C. burnetii
by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5. Measuring in vitro internalization of [
35
S]methionine-[
35
S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake
C. burnetii
was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable
C. burnetii
was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.