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PubMed2006

High throughput modular chambers for rapid evaluation of anesthetic sensitivity.

BACKGROUND: Anesthetic sensitivity is determined by the interaction of multiple genes. Hence, a dissection of genetic contributors would be aided by precise and high throughput behavioral screens. Traditionally, anesthetic phenotyping has addressed only induction of anesthesia, evaluated with dose-response curves, while ignoring potentially important data on emergence from anesthesia. METHODS: We designed and built a controlled environment apparatus to permit rapid phenotyping of twenty-four mice simultaneously. We used the loss of righting reflex to indicate anesthetic-induced unconsciousness. After fitting the data to a sigmoidal dose-response curve with variable slope, we calculated the MAC(LORR) (EC50), the Hill coefficient, and the 95% confidence intervals bracketing these values. Upon termination of the anesthetic, Emergence timeRR was determined and expressed as the mean +/- standard error for each inhaled anesthetic. RESULTS: In agreement with several previously published reports we find that the MAC(LORR) of halothane, isoflurane, and sevoflurane in 8-12 week old C57BL/6J mice is 0.79% (95% confidence interval = 0.78-0.79%), 0.91% (95% confidence interval = 0.90-0.93%), and 1.96% (95% confidence interval = 1.94-1.97%), respectively. Hill coefficients for halothane, isoflurane, and sevoflurane are 24.7 (95% confidence interval = 19.8-29.7%), 19.2 (95% confidence interval = 14.0-24.3%), and 33.1 (95% confidence interval = 27.3-38.8%), respectively. After roughly 2.5 MAC(LORR) x hr exposures, mice take 16.00 +/- 1.07, 6.19 +/- 0.32, and 2.15 +/- 0.12 minutes to emerge from halothane, isoflurane, and sevoflurane, respectively. CONCLUSION: This system enabled assessment of inhaled anesthetic responsiveness with a higher precision than that previously reported. It is broadly adaptable for delivering an inhaled therapeutic (or toxin) to a population while monitoring its vital signs, motor reflexes, and providing precise control over environmental conditions. This system is also amenable to full automation. Data presented in this manuscript prove the utility of the controlled environment chambers and should allow for subsequent phenotyping of mice with targeted mutations that are expected to alter sensitivity to induction or emergence from anesthesia.

Journal Article↗
PubMed2002

Reaction to frustration in high and low feather pecking laying hens.

Reaction to frustration of high (HFP) and low feather pecking (LFP) laying hens was investigated. From a HFP- and a LFP-line five birds with a HFP- and five birds with a LFP-phenotype were selected. Birds from the HFP-line were expected to show more key pecking and covered feeder pecking during frustration than birds from the LFP-line. When a bunch of feathers was presented, birds with a HFP-phenotype were expected to redirect their pecks at the bunch. Birds were trained to peck a key for a food reward in an automated Skinnerbox and subjected to two sessions: a control session, where food was available, and a frustration session, where the feeder was covered with Perspex. These two sessions were repeated in the presence of a bunch of feathers. Unexpectedly, birds from the LFP-line had a stronger reaction to frustration than birds from the HFP-line, expressed in pecking behaviour. When a bunch of feathers was offered, birds with a HFP-phenotype did not show more bunch pecking during frustration than birds with a LFP-phenotype.

Journal Article↗
PubMed1998

Differential genome analysis applied to the species-specific features of Helicobacter pylori.

We introduce a simple and rapid strategy to identify genes that are responsible for species-specific phenotypes. The genome of a species that has a specific phenotype is compared with at least one, closely related, species that lacks this phenotype. Homologous genes that are shared among the species compared are identified and discarded from the list of candidates for species-specific genes. The process is automated and rapidly yields a small subset of the genome that likely contains genes responsible for the species-specific features. Functions are assigned to the genes, and dubious annotations are filtered out. Information is extracted not only from the presence of genes, but also from their absence with respect to known phenotypes. We have applied the technique to identify a set of species-specific genes in Helicobacter pylori by comparing it with its closest relatives for which complete genome sequences are available, Haemophilus influenzae and Escherichia coli. Of the genes of this set for which functional features can be obtained, a large fraction (63%, 123 proteins) is (potentially) involved in H. pylori's interaction with its host. We hypothesize that a family of outer membrane proteins is critical for the ability of H. pylori to colonize host cells in highly acidic environments.

Amino Acid Sequence↗
PubMed1992

Contamination of flexible fiberoptic bronchoscopes with Mycobacterium chelonae linked to an automated bronchoscope disinfection machine.

Between December 5, 1989, and September 25, 1990, Mycobacterium chelonae was isolated from endoscopic or bronchial washings in 14 patients on a single clinical service. A phenotypically unique strain of M. chelonae subspecies abscessus that was highly resistant to cefoxitin (MIC greater than 256 micrograms/ml) and different from 13 control isolates of M. chelonae recovered elsewhere in the hospital was identified in all these patients and the rinse water from the bronchoscope disinfecting machine. None of the outbreak patients had evidence of invasive M. chelonae disease. Aggressive infection control measures on the disinfecting machine, including use of sterile water in the wash and rinse cycles, increasing the 2% alkaline glutaraldehyde exposure time, frequent replacement of the glutaraldehyde, and disinfection of the machine, failed to eradicate the M. chelonae, presumably because of the presence of a biofilm inside the machine. Rinsing the scopes with 70% alcohol after automated disinfection eliminated the outbreak strain. This study demonstrates that automated bronchoscope disinfecting machines may become heavily contaminated with mycobacteria that resist usual disinfection, resulting in a source of bronchoscope contamination.

Bronchoscopes↗
PubMed2025

Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy.

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that these strains have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.

Journal Article↗
PubMed2025

Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy.

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Since Clade 5 strains are highly prevalent in agricultural animals and a frequent cause of zoonotic infections, these strains may have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches.

Clostridioides difficile↗
PubMed1993

A microplate system for ABO and Rh(D) blood grouping.

A microplate system for performing ABO and Rh(D) blood group determinations with a Kemble Kemtek 1000SP liquid handling processor, an Anthos 2001 microplate reader, an IBM Personal System 2 microcomputer, and Sanguin Forma software is described. The performance of this Kemble/Anthos/IBM/Sanguin microplate system for ABO and D grouping was evaluated by testing 10,042 routine blood donor samples in parallel with the forward-grouping channels of a Technicon AutoAnalyzer blood grouping system. Manual techniques were used to perform ABO reverse groupings and to resolve all ABO forward- and reverse-grouping discrepancies. D-negative test results were investigated and confirmed manually by the indirect antiglobulin test. Of the 10,042 samples tested, 97.3 percent were grouped correctly. There were 266 samples whose results were flagged as no type determined, of which 124 were ABO tests and 142 were Rh tests. In addition, 30 weak D samples missed by both automated systems were detected by manual indirect antiglobulin test. The system is flexible and easy to maintain and operate.

ABO Blood-Group System↗
PubMed2001

A neural classifier enabling high-throughput topological analysis of lymphocytes in tissue sections.

A neural cell detection system (NCDS) for the automatic quantitation of fluorescent lymphocytes in tissue sections is presented in this paper. The system acquires visual knowledge from a set of training cell-image patches selected by a user. The trained system evaluates an image in 2 min calculating: the number, the positions, and the phenotypes of the fluorescent cells. For validation, the NCDS learning performance was tested by cross validation on digitized images of tissue sections obtained from inherently different types of tissue: diagnostic tissue sections across the human tonsil and across an inflammatory lymphocyte infiltrate of the human skeletal muscle. The NCDS detection results were compared with detection results from biomedical experts and were visually evaluated by our most experienced biomedical expert. Although the micrographs were noisy and the fluorescent cells varied in shape and size, the NCDS detected a minimum of 95% of the cells. In contrast, the cellular counts based on visual cell recognition of the experts were inconsistent and largely unreproducible for approximately 80% of the lymphocytes present in a visual field. The data indicate that the NCDS is rapid and delivers highly reproducible results and, therefore, enables high-throughput topological screening of lymphocytes in many types of tissue, e.g., as obtained by routine diagnostic biopsy procedures. High-throughput screening with the NCDS provides the platform for the quantitative analysis of the interrelationship between tissue environment, cellular phenotype, and cellular topology.

Automation↗
PubMed2004

Evaluation of the Uro-Quick, a new rapid automated system, for the detection of well-characterized antibiotic-resistant bacteria.

The Uro-Quick system has been employed to detect antibiotic resistance in genotypically and/or phenotypically well-characterized bacterial species including those that might not be easily identified by routine procedure. In order to achieve full agreement between the antibiotic susceptibility results obtained by the reference method (NCCLS) and the Uro-Quick system, the optimal experimental conditions (inoculum size, time of incubation and antibiotic concentration) for each strain to be used by the automatic system were determined. The shorter time periods for generation of correct susceptibility results were 180 min for ampicillin- and ciprofloxacin-resistant Escherichia coli and for ESBL- and Inhibitor-resistant TEM (IRT)-producing E. coli; 360 min for penicillin-susceptible Streptococcus pneumoniae, as well as for strains with reduced susceptibility to this antibiotic (both intermediate, and resistant isolates). The same time was required to detect erythromycin-resistant pneumococci irrespective of their mechanism of resistance (ribosomal methylation and efflux-mediated), Streptococcus pyogenes exhibiting the three erythromycin-resistance phenotypes (constitutive, inducible and M-type) and Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis and Moraxella morganii refractory to third-generation cephalosporins, aminoglycosides, ciprofloxacin and other classes of antimicrobial agents; 480 min for penicillin-resistant, constitutive and inducible oxacillin-resistant (OXA-R) Staphylococcus aureus and OXA-R Staphylococcus epidermidis. The same period of time was also necessary to find the great majority of drug-resistance exhibited by Pseudomonas aeruginosa. Teicoplanin-resistant Staphylococcus haemolyticus, vancomycin-resistant (VanA, VanB, VanC) high-level aminoglycoside-resistant (HLAR) Enterococcus spp, and imipenem-resistant P. aeruginosa required longer incubation (24 h) to be detected. The results obtained indicate that Uro-Quick might be a reliable and promising instrument for the correct detection of the above antibiotic resistance markers.

Anti-Bacterial Agents↗
PubMed1994

Biochemical phenotypes of Salmonella Livingstone isolated from humans, animals and feedstuffs in Sweden.

Salmonella Livingstone is occasionally isolated from humans, animals and feedstuffs in Sweden. To follow the spread of infection and trace the source of isolates, adequate typing methods are needed. We have developed an automated typing system based on biochemical fingerprinting of bacteria (the PhP system) for typing of different Salmonella serotypes. The system measures the kinetics of various biochemical reactions of bacteria grown in liquid medium in microtiter plates and uses numerical techniques to identify biochemical phenotypes (BPTs) among the tested strains. In the present study we used a set of 16 highly discriminatory tests to differentiate strains of Salmonella of serotype Livingstone and evaluated the system for its discriminatory ability using a collection of 34 unrelated human isolates of S. Livingstone. We also used the system to investigate BPTs of 45 Livingstone strains isolated from animals and feedstuffs in Sweden between 1987 and 1991. Altogether 19 different BPTs were found among human isolate giving a diversity index (Di) of 0.930. In contrast, most strains isolated from animals and feedstuffs in Sweden belonged to 2 dominating BPTs (Di = 0.704). One of these contained 17 strains mainly isolated during 1992 whereas the other contained 18 strains isolated between 1987 and 1991. None of the Swedish human isolates were identical to those of animals and feedstuffs. These findings suggest that 2 different BPTs of Salmonella Livingstone strains are particularly common among animals and feedstuffs in Sweden and that they are not related to human cases of enteritis in this country. We also conclude that biochemical fingerprinting with the PhP system is a reliable and highly discriminatory method for detecting epidemic strains of Salmonella Livingstone.

Animal Feed↗
PubMed2006

Maculopathy due to the R345W substitution in fibulin-3: distinct clinical features, disease variability, and extent of retinal dysfunction.

PURPOSE: To determine (1) clinical features that distinguish maculopathy due to the R345W substitution in fibulin-3 from other forms of inherited or early-onset drusen, (2) the phenotypic variability, and (3) the extent of retinal disease in those with a positive molecular diagnosis. METHODS: Affected individuals underwent ophthalmic examination, digital color fundus photography, fundus autofluorescence (AF) imaging, and psychophysical testing with automated photopic and dark-adapted perimetry and fine matrix mapping. Blood samples were taken for DNA extraction and screening for the R345W mutation in fibulin-3. Patients were subsequently divided into mutation-positive and -negative groups, to compare the identified phenotypic findings in these two sets of subjects. RESULTS: Twenty-nine subjects from 19 families were ascertained with inherited or early-onset drusen. Twenty-four (83%) subjects from 15 families were found to harbor the R345W fibulin-3 mutation. Peripapillary deposition and a radial distribution of macular drusen were consistent, distinguishing signs in the mutation-positive group. Subretinal neovascular membrane (SRNVM) was a rare occurrence, affecting only 1 of 48 eyes, whereas hyperpigmentation and atrophy of the retinal pigment epithelium (RPE) were common in older mutation-positive patients. Increased AF corresponding to the drusen was detected in both the mutation-positive and -negative groups. The phenotype in the group of patients positive for the R345W mutation was extremely variable, with evidence of interocular, intrafamilial, and interfamilial variability in visual loss, natural history, ophthalmoscopic findings, autofluorescence imaging, and psychophysical data. The novel finding of nonpenetrance was observed in a 62-year-old asymptomatic, mutation-positive man. The findings from detailed perimetry performed on a subset of subjects were consistent with the presence of widespread retinal dysfunction not isolated to the macula. CONCLUSIONS: Marked inter- and intrafamilial variation associated with the fibulin-3 R345W mutation in terms of retinal appearance, severity, progression, and nonpenetrance were identified. It was noted that SRNVM is a rare occurrence in R345W fibulin-3 maculopathy. These findings are helpful for advice regarding prognosis and for genetic counseling. The findings established that the presence of peripapillary deposit is highly likely to indicate that a patient carries the R345W mutation.

Adult↗
PubMed2002

Antimicrobial susceptibility test: from bacterial population analysis to therapy.

The statistical methods and parameters commonly used to define bacterial susceptibility to antibiotics in vitro such as MIC(50), linear regression or others, usually lead to a considerable loss of information: they do not take into account the heterogeneity of the bacterial population. In contrast, multivariate data analyses are more adapted to the description of biological systems. In this way, a population of a given bacterial species can be separated into homogenous classes corresponding to the different sensitivity and resistance phenotypes. The applications of this mathematical approach include: (i) a new model for more relevant interpretation of antimicrobial susceptibility test results; (ii) numerical estimation of breakpoints having a known risk; (iii) calibration of a technique relative to a reference technique; (iv) detection of strains with new phenotypes; (v) in vitro evaluation of the activity of new compounds.

Anti-Bacterial Agents↗
PubMed2004

Evaluation of different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureus.

To evaluate different methods for their abilities to detect methicillin resistance in small-colony variants (SCVs) of Staphylococcus aureus, 11 different methicillin-resistant S. aureus (MRSA) clones with the SCV phenotype were used in this study. The slow growth of SCVs often makes testing by disk diffusion or by automated methods invalid. Only detection of the mecA gene by PCR and the MRSA-Screen latex agglutination test using a higher colony number were shown to be reliable methods to rapidly detect methicillin resistance in these variants.

Genetic Variation↗
PubMed1994

Haemophilia A diagnosis by automated fluorescent DNA detection of ten factor VIII intron 13 dinucleotide repeat alleles.

Haemophilia A is a recessive X linked bleeding disorder caused by deficiency or functional abnormality of coagulation factor VIII. This disease usually has no visible phenotype in female carriers; hence, great efforts are made to offer all haemophilia A families accurate carrier diagnosis. Significant progress in this direction was made with the identification of the intron 13 variable number tandem repeat (VNTR), which is hitherto the most informative single marker within the factor VIII gene. The authors have established intron 13 VNTR detection in their laboratory by adapting its analysis to an automated sequencer using different primers of which one is fluorescent dye labelled. With this method, which is more rapid and convenient than that originally described, 67 haemophilia A families of German origin were screened and two new alleles (alleles 17 and 25) were identified. The informativeness of the VNTR in these families based on the patients maternal X chromosomes (134) is about 67%.

Alleles↗
PubMedDate not supplied

Partial automation of database processing of simulation outputs from L-systems models of plant morphogenesis.

Models of plant architecture allow us to explore how genotype environment interactions effect the development of plant phenotypes. Such models generate masses of data organised in complex hierarchies. This paper presents a generic system for creating and automatically populating a relational database from data generated by the widely used L-system approach to modelling plant morphogenesis. Techniques from compiler technology are applied to generate attributes (new fields) in the database, to simplify query development for the recursively-structured branching relationship. Use of biological terminology in an interactive query builder contributes towards making the system biologist-friendly.

Automation↗
PubMed2004

[Accuracy of four agar diffusion methods and the Vitek 2 automated system for the detection of the methicillin resistance in coagulase negative staphylococci].

AIM OF THE STUDY: Technical difficulties may occur in the detection of methicillin resistance in coagulase negative staphylococci. Phenotypic methods, such as disk diffusion testing, sometimes fail to detect methicillin resistance because of its poor expression. Four of these methods were compared with the VITEK 2 system. MATERIALS AND METHODS: The accuracy of the VITEK 2 system and the agar diffusion methods according to the current CASFM and NCCLS guidelines were assessed for methicillin susceptibility testing of 70 coagulase negative staphylococci isolates harboring the mecA gene for which the detection of the methicillin resistance was particularly difficult: the CASFM recommendations applied in 1998 failed to categorize them as methicillin-resistant. RESULTS: Among the different methods currently suggested by the CASFM, those using a heavy inoculum and a hypersaline medium give better results, but they remain however less efficient than the NCCLS recommendations testing (sensitivity: 71-86% vs. 94-94% respectively). The VITEK 2 was more efficient (sensitivity: 91%) than the disk diffusion methods recommended by the CASFM and slightly less efficient than the method recommended by the NCCLS. CONCLUSION: The VITEK 2 automated system, compared with the disk diffusion methods, showed adequate accuracy for detection of oxacillin resistance in coagulase negative staphylococci.

Coagulase↗
PubMed2005

Methods to record circadian rhythm wheel running activity in mice.

Forward genetic approaches (phenotype to gene) are powerful methods to identify mouse circadian clock components. The success of these approaches, however, is highly dependent on the quality of the phenotype--specifically, the ability to measure circadian rhythms in individual mice. This article outlines the factors necessary to measure mouse circadian rhythms, including choice of mouse strain, facilities and equipment design and construction, experimental design, high-throughput methods, and finally methods for data analysis.

Animals↗