bluuebunny/crossref_metadata_2025 huggingface dataset:
O6  The occurrence and cause of extra‐oral halitosis
Authors: Tangerman A , Winkel EG
Published: March 2005

Objective  Oral malodor is now generally accepted to be the main cause (about 90%) of bad breath. Bacterial formation of the volatile sulfur compounds hydrogen sulfide (H2S) and methylmercaptan (MM, CH3SH) within the oral cavity is the main cause of oral malodor. The objective was to study the occurrence and cause of the less well‐known extra‐oral halitosis.

Methods  We investigated the mouth‐ and nose‐breath of 59 subjects complaining of bad breath and visiting our bad breath clinic. The methods used were gas chromatography and organoleptic scoring (OLS).

Results  Six of 59 patients (10 %) were found to have extra‐oral halitosis. The concentrations of H2S and MM in mouth‐breath of patients with extra‐oral halitosis were very low and below critical odorous levels. H2S and MM were absent in nose‐breath of all patients. The concentration of dimethylsulfide (DMS, CH3SCH3) in mouth‐ and nose‐breath in the six patients with extra‐oral halitosis amounted to 1.57 ± 1.31 (s.d.) and 1.64 ± 1.29 nmol l−1 (range: 0.50–4.50), respectively, compared to 0.29 ± 0.19 and 0.21 ± 0.12 nmol l−1, respectively, in patients with oral malodor. The critical odorous concentration of DMS is about 0.5 nmol l−1. Simulated DMS gas‐mixtures had the same smell as observed in the extra‐oral halitosis patients. In these six patients good to perfect correlations were found between the OLS of mouth‐ and nose‐breath (simple correlation coefficient r = 0.840, P < 0.05), between mouth‐OLS and the concentration of DMS in mouth‐breath (r = 0.794, P < 0.05) and in nose‐breath (r = 0.824, P < 0.05) and between nose‐OLS and the concentration of DMS in mouth‐breath (r = 0.963, P < 0.001) and in nose‐breath (r = 0.955, P < 0.001). A perfect correlation was obtained between mouth‐ and nose‐DMS (r = 0.994, P < 0.001), indicating that DMS does not originate in the mouth . The origin of DMS must be found elsewhere, most probably in the blood.

Conclusions  About 5–10% of patients with chronic halitosis has extra‐oral halitosis. In all patients with extra‐oral halitosis seen thus far, DMS was always the cause of bad breath. Other causes known, such as trimethylamine in the fish‐odor syndrome, are very rare.
P39  Evaluation of patient oriented outcome using SF‐36 in halitosis therapy
Authors: Kishi M , Abe A , Yonemitsu M
Published: March 2005

Objective  To evaluate the patient oriented outcome of medical interventions, Medical Outcome Study Short Form‐36 (SF‐36) is a widely used, self‐administered questionnaire. The SF‐36 has eight dimensions of health status consisting of Physical Function (PF), Role‐ Physical (RP), Bodily Pain (BP), General Health (GH), Vitality (VT), SF (Social Function), Role‐ Emotion (RE) and Mental Health (MH). Furthermore, each dimensional score and two summary scores about physical and mental regions can be calculated by the algorithm. The aim of this study was to examine the application of SF‐36 to the clinical parameter of halitosis.

Subjects and methods  Seventy patients visiting our special clinic for halitosis were the subjects. In this study, we defined the healed persons as follows. They received our therapy two or more times, and the duration was over 2 months. After our therapy, they declared that the oral malodor of themselves could not bother them. In addition, they accepted to receive the general dental check‐up regularly after the initial therapeutic period. The SF‐36 was filled by the subjects at the first visit of our clinic and the first time of the regular check‐up. The content of our therapy was mainly involved measurement of oral malodor, supporting for oral self‐care, professional oral cleaning and counseling. All management concerning SF‐36 was performed according to the manual of SF‐36, version 1.2, Japanese edition.

Results and discussion  Among all 70 subjects, GH, VT, SF, RE, MH and mental summary scores were significantly lower than national averages. Among the healed persons (n = 17), only SF score significantly rose after receiving our therapy. These results suggest that the consciousness of the improvement in social function could be an important outcome of halitosis therapy. In conclusion, measuring health related QOL using SF‐36 might be useful for evaluating the efficacy of halitosis therapy.
P40  Measurement of VSC and organoleptic scores and subjective patients’ opinion about halitosis – a questionnaire study
Authors: Iwanicka‐Grzegorek E , Michalik J , Kepa J , Wierzbicka M , Aleksinski M , Pierzynowska E
Published: March 2005

Objectives  The aim of the study was to evaluate the level of interest in fetor ex ore among respondents, patients of the Department of Conservative Dentistry in Warsaw.

Methods  A questionnaire was completed by 295 patients, 202 female and 93 male aged 18–74 (avg 38.73). Each person was also examined for organoleptic score and VSC by halimeter. Halitosis was diagnosed if the average level of VSC measured by halimeter was ≥125 ppb and the organoleptic measurement using a 5‐point scale was ≥2. Statistical analysis was performed using Chi square test.

Results  Incidence of halitosis was greatest in age ranges 25–34 (29.68%) and 45–54 years (24.52%). People from these age groups are more careful about oral heath, so they are more likely to be interested in checking for halitosis. Among 295 respondents 25.42% often had a problem with bad breath, 39.66% sometimes, and 34.91% rarely or never. Persons with diagnosed halitosis frequently reported having a problem with bad breath (47.73%), and only 5.76% persons with halitosis did not notice this problem (P < 0.001). Among 76 healthy persons five (6.6%) reported having a problem with halitosis (P < 0.001). Clinical tests for halitosis allowed a diagnosis of halitophobia in four cases. Respondents noticed a problem with bad breath in their relatives in 46.0%. The frequency of malodor observed in relatives of patients with halitosis was similar to that in relatives of healthy subjects (P = 0.58). No correlation was found between cigarette smoking and a diagnosis of halitosis (P = 0.22).

Conclusions  A statistically significant correlation was found between clinical diagnosis of halitosis and VSC level by halimeter, organoleptic measurement and subjective patients’ opinion.
P41  Tongue cleaning habits in Italians: results of a survey in a sample population
Authors: Abati S , Ferrieri G , Strohmenger L
Published: March 2005

Background and objectives  Mechanical cleaning of the tongue is suggested as an effective method for reducing cultivable flora of the tongue and teeth and decreasing oral malodor. The aim of this study was to evaluate the general attitude, knowledge and behaviour of a representative sample of Italian population regarding the hygienic tongue procedures.

Methods  The survey has been conducted in January and February 2003 through structured phone interviews using CATI (computer aided telephone interviews) methods by EuriskoOmnibus, a specialized agency, on commission by GlaxoSmithKline Consumer Healthcare S.p.A.‐Italy. The sample has been randomly chosen among urban and rural residents from age 15 onwards with a nationwide distribution in proportion to the last demoskopic feature of general Italian population. Relevant data has been processed with MSExcel and StatView software in an Apple G4 Powerbook PC.

Results  The sample is composed by 2000 individuals, 52% females and 48% males. Of these, 6% have an academic degree, 23% high school and 71% lower middle school Over one‐third (39%) of Italians interviewed declared to perform specifically tongue cleaning: 40% of them every time they perform oral hygiene twofold or more daily, 24% once a day and 36% less than daily; the tongue cleaning habits are more diffuse in younger people. Seventy‐one per cent of tongue cleaners perform the procedure with an ordinary toothbrush, 22% declared to clean tongue with an oral rinse and only 4% utilize specific tongue instruments. In our sample main reasons for tongue cleaning practices were reaching a better oral hygiene (60%) and fighting bad breath (25%).

Conclusions  In Italians tongue cleaning is more diffuse than expected but the reasons are not principally related to the reduction of halitosis.
O7  The treatment of oral malodor
Authors: Kazor CE , Loesche WJ
Published: March 2005

Objective  Because the increase in the bacterial load on the tongue is paramount in the etiology of malodor, we sought to reduce this load by debriding the tongue in combination with short‐term usage of antimicrobial mouthrinses.

Methods  Subjects with oral malodor had an OR score >2, had whole mouth volatile sulfur (VSCs) levels ≥200 ppb (Halimeter®), and had a positive tongue BANA test. The subjects were given a tongue scraper and a 16 oz bottle of mouthrinse (Listerine® or Ther‐a‐sol®, single blind design). They were instructed to debride their dorsal tongue surface, to brush and floss the teeth, to rinse with 1 oz of the mouthrinse for 1 min, instructed not to eat or drink for 2–3 h thereafter, and to repeat the protocol in the morning and evening for 7 days. After 1 week all malodor measurements were repeated. The patients were then given a zinc containing tongue gel and instructed to debride the tongue surface, repeating this procedure twice a day.

Results and discussion  Thirty‐two subjects entered this program, 31 returned 1 week later, and 20 returned for a follow‐up examination (average of 37 weeks). The OR score decreased from 2.8 to 0.9 after 1 week of treatment, and to 0.6 for those who returned for the follow‐up. The VSCs decreased from 339 ppb prior to treatment to 120 ppb after 1 week and to 53 ppb 37 weeks later. Eighty‐one percent of the tongues were BANA positive prior to treatment, 16% after 1 week and 0% after 37 weeks. The microbial flora changed from one dominated by asaccharolytic species to one dominated by saccharolytic species such as S. salivarius.

Conclusions  This report describes a long‐term beneficial effect of treatment on oral malodor.
O8  Impact of periodontal therapy (including antiseptics) on tongue coating and malodour
Authors: Van Steenberghe D , Zhao H , Soers C , Pauwels M , Quirynen M
Published: March 2005

Background  Bad breath is often caused by periodontitis and/or tongue coating. This study aimed to follow, over a 6 months period, the impact of periodontal therapy on several parameters linked with oral malodour.

Methods  In this double‐blind, randomized, long‐term, parallel study 45 moderate periodontitis patients without obvious tongue coating were enrolled. Besides a ‘one‐stage, full‐mouth’ disinfection and oral hygiene improvement (including tongue scraping), patients were instructed to rinse with one of the following products (randomly allocated): CHX 0.2% + alcohol (Corsodyl®), CHX 0.05% + CPC 0.05% and no alcohol (Perio.Aid®Maintenance, a new formulation), or the placebo of the latter for 6 months. At baseline and after 3 and 6 months, a serious of parameters was recorded including VSC rating (Halimeter), tongue coating and an estimation of the microbial load (anterior & posterior part tongue, saliva, supragingival plaque).

Results  Even though the initial VSC values were not high (±90, only 18 patients >100), significant (P < 0.05) reductions could be recorded in the CHX and CHX+CPC group, but only to a minor extent in the placebo group. Tongue scraping resulted in a significant reduction (P ≤ 0.05) of the coating up to month 6 in the placebo and CHX+CPC group, but not in the CHX group. The microbial changes in the placebo group were never significant (≤0.3 log values), even though the tongue had been scraped daily. The CHX and CHX‐CPC group showed, in comparison to baseline, significant (P < 0.001) reductions in the number of anaerobic species in the supra gingival plaque, in the saliva and on the anterior part of the tongue. For the posterior part of the tongue the microbial changes remained ≤0.3 log values.

Conclusions  The results of this study indicate that, in patients with moderate periodontitis, periodontal therapy including tongue scraping did not have a significant effect on the microbial load of the tongue or the VSC level, except when combined with a mouthrinse.
Workshop 1  Odor measurements using human judges (including judge training and standardization)
Authors: Nachnami Sushma
Published: March 2005

This group consisted of participants from various academic institutes and industry:

Most of the academic research groups had no formal training program in place. Several of the group members had worked with John Greenman's group to train and standardize. Also, a couple of academicians did set up some type of training where they at least worked together smelling human breath to train and calibrate before they began seeing patients. However the industry had elaborate training programs in place.

Samples used by industry for training were mostly garlic and onions; some of the groups used scratch & sniff tests or essential oils.

Organoleptic judges should have training documentation after completion of training. This should be recommended to the ADA.

Airborne infections:  Concern about sniffing patients in close proximity was raised. Some people in industry monitor their judges closely by conducting medical exams on an ongoing basis. Medical histories of the patients should be looked at closely. Universal precaution laws should be followed closely.

How to sniff:  Different methods were discussed, ‘blinding’ helps. Different ways of sniffing – some used glass tube, breathing out, e.g. counting out loud to twenty or saying ha ha ha etc.

Descriptors should be provided to the group for training as it is difficult for organoleptic judges to come up with appropriate phrases. While most of the academic groups trained directly with subjects, several academic groups reported developing training programs based on ASTM standards. The people from Industry all used ASTM standards. Certification was provided after 1 year of training with subjects.
Life at Depth:
Photobacterium profundum
Genome Sequence and Expression Analysis
Authors: Vezzi A. , Campanaro S. , D'Angelo M. , Simonato F. , Vitulo N. , Lauro F. M. , Cestaro A. , Malacrida G. , Simionati B. , Cannata N. , Romualdi C. , Bartlett D. H. , Valle G.
Published: March 2005

Deep-sea life requires adaptation to high pressure, an extreme yet common condition given that oceans cover 70% of Earth's surface and have an average depth of 3800 meters. Survival at such depths requires specific adaptation but, compared with other extreme conditions, high pressure has received little attention. Recently,
Photobacterium profundum
strain SS9 has been adopted as a model for piezophily. Here we report its genome sequence (6.4 megabase pairs) and transcriptome analysis. The results provide a first glimpse into the molecular basis for life in the largest portion of the biosphere, revealing high metabolic versatility.
Requirement for Caspase-8 in NF-κB Activation by Antigen Receptor
Authors: Su Helen , Bidère Nicolas , Zheng Lixin , Cubre Alan , Sakai Keiko , Dale Janet , Salmena Leonardo , Hakem Razqallah , Straus Stephen , Lenardo Michael
Published: March 2005

Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor κB (NF-κB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the αβ complex of the inhibitor of NF-κB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKα, β complex, its activation, and the nuclear translocation of NF-κB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.
Supramolecular Assembly of Amelogenin Nanospheres into Birefringent Microribbons
Authors: Du Chang , Falini Giuseppe , Fermani Simona , Abbott Christopher , Moradian-Oldak Janet
Published: March 2005

Although both tooth enamel and bone are composed of organized assemblies of carbonated apatite crystals, enamel is unusual in that it does not contain collagen nor does it remodel. Self-assembly of amelogenin protein into nanospheres has been recognized as a key factor in controlling the oriented and elongated growth of carbonated apatite crystals during dental enamel biomineralization. We report the in vitro formation of birefringent microribbon structures that were generated through the supramolecular assembly of amelogenin nanospheres. These microribbons have diffraction patterns that indicate a periodic structure of crystalline units along the long axis. The growth of apatite crystals orientated along the
c
axis and parallel to the long axes of the microribbons was observed in vitro. The linear arrays (chains) of nanospheres observed as intermediate states before the microribbon formation give an important indication as to the function of amelogenin in controlling the oriented growth of apatite crystals during enamel mineralization.
A Functional Dosage Compensation Complex Required for Male Killing in
Drosophila
Authors: Veneti Zoe , Bentley Joanna K. , Koana Takao , Braig Henk R. , Hurst Gregory D. D.
Published: March 2005

Bacteria that selectively kill males (“male-killers”) were first characterized more than 50 years ago in
Drosophila
and have proved to be common in insects. However, the mechanism by which sex specificity of virulence is achieved has remained unknown. We tested the ability of
Spiroplasma poulsonii
to kill
Drosophila melanogaster
males carrying mutations in genes that encode the dosage compensation complex. The bacterium failed to kill males lacking any of the five protein components of the complex.
Anthropologists in the Lab
Authors: Haseltine William A.
Published: March 2005

A Machine to Make a Future
Biotech Chronicles.
By Paul Rabinow and Talia Dan-Cohen
. Princeton University Press, Princeton, NJ, 2004. 204 pp. $24.95, £15.95. ISBN 0-691-12050-1.

The authors, anthropologists who focus their attention on the current practices of science, offer a behind-the-scenes look at the intersection of genomics, medicine, and commerce that is focused on Celera Diagnostics
Differential Degradation of Nonylphenol Isomers by
Sphingomonas xenophaga
Bayram
Authors: Gabriel Frédéric L. P. , Giger Walter , Guenther Klaus , Kohler Hans-Peter E.
Published: March 2005

Sphingomonas xenophaga
Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-
n
-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C
9
alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-
n
-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-
n
-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.
Development of Procedures for Direct Extraction of
Cryptosporidium
DNA from Water Concentrates and for Relief of PCR Inhibitors
Authors: Jiang Jianlin , Alderisio Kerri A. , Singh Ajaib , Xiao Lihua
Published: March 2005

Extraction of high-quality DNA is a key step in PCR detection of
Cryptosporidium
and other pathogens in environmental samples. Currently,
Cryptosporidium
oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of
Cryptosporidium
with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/μl or 25 ng of T4 gene 32 protein/μl to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.
Validation of a Flour-Free Model Dough System for Throughput Studies of Baker's Yeast
Authors: Panadero Joaquin , Randez-Gil Francisca , Prieto Jose Antonio
Published: March 2005

Evaluation of gene expression in baker's yeast requires the extraction and collection of pure samples of RNA. However, in bread dough this task is difficult due to the complex composition of the system. We found that a liquid model system can be used to analyze the transcriptional response of industrial strains in dough with a high sugar content. The production levels of CO
2
and glycerol by two commercial strains in liquid and flour-based doughs were correlated. We extracted total RNA from both a liquid and a flour-based dough. We used Northern blotting to analyze mRNA levels of three stress marker genes,
HSP26
,
GPD1
, and
ENA1
, and 10 genes in different metabolic subcategories. All 13 genes had the same transcriptional profile in both systems. Hence, the model appears to effectively mimic the environment encountered by baker's yeast in high-sugar dough. The liquid dough can be used to help understand the connections between technological traits and biological functions and to facilitate studies of gene expression under commercially important, but experimentally intractable, conditions.
Induction of Shiga Toxin-Converting Prophage inEscherichia coliby High Hydrostatic Pressure
Authors: Aertsen Abram , Faster David , Michiels Chris W.
Published: March 2005

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response inEscherichia coliMG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treatingE. colilysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producingE. colistrains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa inE. coliMG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.
Effect of Heat, Acidification, and Chlorination on
Salmonella enterica
Serovar Typhimurium Cells in a Biofilm Formed at the Air-Liquid Interface
Authors: Scher Keren , Romling Ute , Yaron Sima
Published: March 2005

Bacterial biofilms have great significance for public health, since biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents and treatments. To date most attention has focused on biofilms that arise from the colonization of solid-liquid or solid-air interfaces. It is of interest that colonization of the interface between air and liquid, which can be selectively advantageous for aerobic or facultative aerobic bacteria, has been rarely studied, although it may present a major problem in industrial aquatic systems. In this work we investigated the role of a biofilm at the interface between air and liquid (pellicle) in the susceptibility of
Salmonella enterica
serovar Typhimurium to stress conditions. For a control we used a mutant that had lost its ability to synthesize cellulose and thin aggregative fimbriae and thus did not produce the pellicle. Resistance of bacteria from the pellicle to heat, acidification, and chlorination was compared to resistance of planktonic cells from the logarithmic and stationary phases of growth. Pellicle cells were significantly more resistant to chlorination, and thus the surrounding matrix conferred protection against the reactive sodium hypochlorite. However, the stress management of pellicle cells in response to heat and low pH was not enhanced compared to that of stationary-phase cells. A long-period of incubation resulted in endogenous hydrolysis of the pellicle matrix. This phenomenon provides a potential new approach to combat microbial cells in biofilms.
Mycobacterium aviumSubspeciesparatuberculosisCultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic
Authors: Ayele Wuhib Y. , Svastova Petra , Roubal Petr , Bartos Milan , Pavlik Ivo
Published: March 2005

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinicalMycobacterium aviumsubsp.paratuberculosisinfection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900PCR results.M. aviumsubsp.paratuberculosiswas cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk.M. aviumsubsp.paratuberculosiswas also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive forM. aviumsubsp.paratuberculosis.These findings confirm thatM. aviumsubsp.paratuberculosisis present in raw milk from subclinically infected dairy cows. The culture ofM. aviumsubsp.paratuberculosisin the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.
Heterologous Production, Assembly, and Secretion of a Minicellulosome by
Clostridium acetobutylicum
ATCC 824
Authors: Mingardon Florence , Perret Stéphanie , Bélaïch Anne , Tardif Chantal , Bélaïch Jean-Pierre , Fierobe Henri-Pierre
Published: March 2005

The gene
man5K
encoding the mannanase Man5K from
Clostridium cellulolyticum
was cloned alone or as an operon with the gene
cipC1
encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic
Clostridium acetobutylicum
. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter P

thl

. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only
man5K
, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When
man5K
was coexpressed with
cipC1
in
C. acetobutylicum
, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that
C. acetobutylicum
is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.
Analyses of Livestock Production, Waste Storage, and Pathogen Levels and Prevalences in Farm Manures
Authors: Hutchison M. L. , Walters L. D. , Avery S. M. , Munro F. , Moore A.
Published: March 2005

Survey results describing the levels and prevalences of zoonotic agents in 1,549 livestock waste samples were analyzed for significance with livestock husbandry and farm waste management practices. Statistical analyses of survey data showed that livestock groups containing calves of <3 months of age, piglets, or lambs had higher prevalences and levels ofCampylobacterspp. andEscherichia coliO157 in their wastes. Younger calves that were still receiving milk, however, had significantly lower levels and prevalence ofE. coliO157. Furthermore, when wastes contained any form of bedding, they had lowered prevalences and levels of both pathogenicListeriaspp. andCampylobacter spp. Livestock wastes generated by stock consuming a diet composed principally of grass were less likely to harborE. coliO157 orSalmonellaspp. Stocking density did not appear to influence either the levels or prevalences of bacterial pathogens. Significant seasonal differences in prevalences were detected in cattle wastes;Listeriaspp. were more likely to be isolated in March to June, andE. coliO157 was more likely to be found in May and June. Factors such as livestock diet and age also had significant influence on the levels and prevalences of some zoonotic agents in livestock wastes. A number of the correlations identified could be used as the basis of a best-practice disposal document for farmers, thereby lowering the microbiological risks associated with applying manures of contaminated livestock to land.
Degradation of 1,4-Dioxane and Cyclic Ethers by an Isolated Fungus
Authors: Nakamiya Kunichika , Hashimoto Syunji , Ito Hiroyasu , Edmonds John S. , Morita Masatoshi
Published: March 2005

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as
Cordyceps sinensis
from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-
d
8
and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.
Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils
Authors: Wang Ya-Juan , Leadbetter Jared Renton
Published: March 2005

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations,
14
C-labeled acyl-HSLs were mineralized to
14
CO
2
rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent

K
m

of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol · h
−1
 · g of fresh weight soil
−1
. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO
2
but that the two processes were well coupled kinetically. A most probable number of 4.6 × 10
5
cells · g of turf soil
−1
degraded physiologically relevant amounts of hexanoyl-[1-
14
C]HSL to
14
CO
2
. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems.
Rhizobial 16S rRNA and
dnaK
Genes: Mosaicism and the Uncertain Phylogenetic Placement of
Rhizobium galegae
Authors: Eardly B. D. , Nour S. M. , van Berkum P. , Selander R. K.
Published: March 2005

The phylogenetic relatedness among 12 agriculturally important species in the order
Rhizobiales
was estimated by comparative 16S rRNA and
dnaK
sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of
Mesorhizobium loti
and
Mesorhizobium ciceri
, and the other group consisted of
Agrobacterium rhizogenes
,
Rhizobium tropici
,
Rhizobium etli
, and
Rhizobium leguminosarum
. Although bootstrap support for the placement of the remaining six species varied,
A. tumefaciens
,
Agrobacterium rubi
, and
Agrobacterium vitis
were consistently associated in the same subcluster. The three other species included
Rhizobium galegae
,
Sinorhizobium meliloti
, and
Brucella ovis
. Among these, the placement of
R. galegae
was the least consistent, in that it was placed flanking the
A. rhizogenes
-
Rhizobium
cluster in the
dnaK
nucleotide sequence trees, while it was placed with the other three
Agrobacterium
species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of
R. galegae
, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between
R. galegae
and certain other species, suggesting that the
R. galegae
genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of
R. galegae
, and they also illustrate a potential pitfall in the use of partial sequences for species identification.
Fingerprinting of
Bacillus thuringiensis
Type Strains and Isolates by Using
Bacillus cereus
Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis
Authors: Reyes-Ramirez Arturo , Ibarra Jorge E.
Published: March 2005

A total of 119
Bacillus thuringiensis
strains (83 type strains and 26 native isolates), as well as five
B. cereus
group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six
B. cereus
group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive
B. cereus
(Bc)-Rep-PCR pattern, except for
B. thuringiensis
serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the
B. thuringiensis
type strain. The
B. cereus
group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify
B. thuringiensis
serotypes, including nonflagellar and self-agglutinated strains.
The Doubly Phosphorylated Form of HPr, HPr(Ser-P)(His∼P), Is Abundant in Exponentially Growing Cells ofStreptococcus thermophilusand Phosphorylates the Lactose Transporter LacS as Efficiently as HPr(His∼P)
Authors: Cochu Armelle , Roy Denis , Vaillancourt Katy , LeMay Jean-Dominique , Casabon Israël , Frenette Michel , Moineau Sylvain , Vadeboncoeur Christian
Published: March 2005

InStreptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(His∼P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr inS. thermophilusATCC 19258 andS. thermophilusSMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinantS. thermophilusATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacSwas reversibly phosphorylated by HPr(Ser-P)(His∼P) at a rate similar to that measured with HPr(His∼P). Sequence analysis of the IIALacSprotein domains from severalS. thermophilusstrains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacSfrom group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacSfrom group II could also be phosphorylated by HPr(His∼P) and HPr(Ser-P)(His∼P), in vitro phosphorylation experiments were conducted with purified proteins fromStreptococcus salivariusATCC 25975, which possesses a IIALacSvery similar to group IIS. thermophilusIIALacS. The results indicated thatS. salivariusIIALacSwas phosphorylated by HPr(Ser-P)(His∼P) at a higher rate than that observed with HPr(His∼P). Our results suggest that the reversible phosphorylation of IIALacSinS. thermophilusis accomplished by HPr(Ser-P)(His∼P) as well as by HPr(His∼P).
16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”
Authors: Loy Alexander , Schulz Claudia , Lücker Sebastian , Schöpfer-Wendels Andreas , Stoecker Kilian , Baranyi Christian , Lehner Angelika , Wagner Michael
Published: March 2005

For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet unculturedZoogloea-,Ferribacterium/Dechloromonas-, andSterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.
Isolation and Characterization of Novel Giant
Stenotrophomonas maltophilia
Phage φSMA5
Authors: Chang Hsiao-Chuan , Chen Chiy-Rong , Lin Juey-Wen , Shen Gwan-Han , Chang Kai-Ming , Tseng Yi-Hsiung , Weng Shu-Fen
Published: March 2005

Stenotrophomonas maltophilia
is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight
S. maltophilia
phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated φSMA5, was further characterized. Electron microscopy showed it resembled
Myoviridae
, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The φSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, φKZ of
Pseudomonas aeruginosa
. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87
S. maltophilia
strains collected in this study, only 61 were susceptible to φSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about
S. maltophilia
phages, φSMA5 appears to be the first reported.
Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations
Authors: Mou Xiaozhen , Moran Mary Ann , Stepanauskas Ramunas , González José M. , Hodson Robert E.
Published: March 2005

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to
Loktanella
,
Oceanicola
, and
Sulfitobacter
(
Roseobacter
lineage, α
-Proteobacteria
);
Caulobacter
(α
-Proteobacteria
); and
Brachymonas
and
Xenophilus
(β
-Proteobacteria
) in the May-02 sample and to
Ketogulonicigenium
(
Roseobacter
lineage, α
-Proteobacteria
) and novel γ
-Proteobacteria
in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.
Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia
Authors: Yu Chang-Ping , Ahuja Rajiv , Sayler Gary , Chu Kung-Hui
Published: March 2005

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, anEscherichia colistrain (gram negative), aPseudomonas fluorescensstrain (gram negative), and aBacillus thuringiensisstrain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.