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Biomedical subjects

S A Carlson

Publications and source records attributed to S A Carlson.

At least 19 recordsLinked to original sources
PubMed2006

Klebsiella to Salmonella gene transfer within rumen protozoa: implications for antibiotic resistance and rumen defaunation.

The rumen has long been thought to be a site of gene transfer for microorganisms. Rumen protozoa (RPz) are active predators of bacteria that can harbor antibiotic resistance genes. In this study, RPz were assessed as sites of gene transfer between two bacterial species, Salmonella and Klebsiella. One Klebsiella isolate carried a plasmid bearing bla(CMY-2), encoding an extended-spectrum beta-lactamase conferring ceftriaxone resistance, while the Salmonella was susceptible to ceftriaxone yet capable of thriving within protozoa. In vitro studies revealed that ceftriaxone-resistant Salmonella could be isolated following co-incubation of Salmonella and Klebsiella with RPz obtained from adult cattle and goats. Ceftriaxone-resistant Salmonella were not recovered in the presence of an inhibitor of protozoa engulfment or when a protozoa-sensitive Salmonella was part of the co-incubation. This transfer event was additionally observed in vitro for protozoa-independent stressors although at a significantly lower frequency. The gene transfer event was related to bacterial conjugation since a conjugation inhibitor, nalidixic acid, perturbed the phenomenon. Ceftriaxone-resistant Salmonella were recovered from calves, sheep, and goats co-challenged with ceftriaxone-resistant Klebsiella and ceftriaxone-sensitive Salmonella. However, the transfer event was not observed in calves and sheep that were defaunated prior to the co-challenge. Moreover, Salmonella transconjugants were isolated from separate bovine in vivo studies involving a Klebsiella donor carrying a plasmid conferring colicin activity while no such transconjugants were obtained from defaunated calves. These results provide an important basis for evaluating and preventing the spread of antibiotic resistance and other selective advantages for pathogens present in ruminants.

Animals↗
PubMed2002

Abomasitis associated with multiple antibiotic resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Salmonella enterica serotype Typhimurium phagetype DT104 is a multiple antibiotic resistant pathogen that has been purported to be more pathogenic than other Salmonella. In this study, we evaluated the possibility that DT104 is the causative agent of veal calf abomasitis observed in four independent outbreaks of salmonellosis. This study was undertaken to determine if the outbreaks might be due to hypervirulent S. enterica serotype Typhimurium phagetype DT104 (DT104) since Salmonella does not usually cause abomasitis. Tissues and fluids from these calves were subjected to bacteriologic culture. Pure Salmonella cultures were then used in bovine challenge experiments. DT104 was identified as the causative agent of abomasitis in calves. Thus, abomasitis is a potential indicator of infection with multiple antibiotic resistant DT104 and adds credence to the apparent hypervirulence of this pathogen.

Abomasum↗
PubMed2002

Early epithelial invasion by Salmonella enterica serovar Typhimurium DT104 in the swine ileum.

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonella serovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion.

Animals↗
PubMed2001

Secretion of a putative cytotoxin in multiple antibiotic resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Salmonella enterica serotype Typhimurium phagetype DT104 (DT104) is a multiple antibiotic-resistant pathogen. DT104 infections have been reported in a multitude of hosts including humans, companion animals, livestock and wildlife. Recently, several isolates of DT104 were recovered from veal calves exhibiting abomasitis, a finding that is inconsistent with classic salmonellosis. One of these isolates was used in murine ligated loop experiments where it was observed that multiresistant DT104 can elaborate a putative cytotoxin. Thus it appears that DT104 has the ability to evade pharmacologic interventions, via antibiotic resistance, and elaborate a toxin that can damage cells.

Animals↗
PubMed2001

Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype typhimurium phage type DT104.

PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 422 veterinary isolates of Salmonella enterica serotype Typhimurium. The identities of extra-integron genes encoding resistance to streptomycin, gentamicin, kanamycin, and apramycin were evaluated. Gentamicin resistance was conferred by the aadB gene. Kanamycin resistance was encoded by either the aphA1-Iab gene or the Kn gene. Apramycin resistance was determined by the aacC4 gene. Analysis of gene distribution did not reveal significant differences with regard to phage type, host species, or region except for the Kn gene, which was found mostly in nonclinical isolates. The data from this study indicate that pentaresistant DT104 does not acquire extra-integron genes in species- or geography-related foci, which supports the hypothesis that clonal expansion is the method of spread of this organism.

Acetyltransferases↗
PubMed2001

Antibiotic resistance in Salmonella enterica serovar Typhimurium exposed to microcin-producing Escherichia coli.

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.

Anti-Bacterial Agents↗
PubMed2000

Augmentation of antibiotic resistance in Salmonella typhimurium DT104 following exposure to penicillin derivatives.

Antibiotic resistance in pathogenic bacteria has been a problem in both developed and developing countries. This problem is especially evident in Salmonella typhimurium, one of the most prevalent foodborne pathogens. While performing in vitro gentamicin protection-based invasion assays, we found that certain isolates of multiresistant S. typhimurium can be 'induced' to exhibit new resistance profiles. That is, bacteria become resistant to a wider range of antibiotics and they also exhibit quantitative increases in MIC values for antibiotics that were part of their pre-induction antibiograms. This 'induction' process involves growing the bacteria to stationary phase in the presence of antibiotics such as ampicillin, amoxicillin or ticarcillin. Since the isolates studied exhibited resistance to ampicillin, amoxicillin and ticarcillin prior to exposing the bacteria to these antibiotics, the observed phenomenon suggests that resistant Salmonella not only have a selective advantage over non-resistant Salmonella but their resistance phenotypes can be accentuated when an inappropriate antibiotic is used therapeutically.

Ampicillin Resistance↗
PubMed2000

Identification of diminished tissue culture invasiveness among multiple antibiotic resistant Salmonella typhimurium DT104.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium further poses a major health concern due to its apparent enhanced ability to acquire multiple antibiotic resistance genes. Currently it is unclear if multiresistant S. typhimurium are more or less pathogenic than non-resistant counterparts. Using an in vitro invasion assay, we evaluated the relative pathogenicity of over 400 multiresistant S. typhimurium isolates. Our studies failed to identify any <<< >>> isolates. However, we identified 12 isolates exhibiting invasive phenotypes that were constrained relative to controls. These <<< >>> strains were found in a variety of phagetypes all possessing at least a hexaresistant profile. Further studies revealed that the alterations in invasion were not due to changes in adherence. Limited studies exploring in vivo virulence revealed a mildly decreased ability to cause murine lethality for the hypoinvasive strain examined. These results indicate that the ability to cause disease is not increased but is rather mildly attenuated for certain isolates of multiresistant S. typhimurium.

Animals↗
PubMed2000

Evaluation of invasion-conferring genotypes and antibiotic-induced hyperinvasive phenotypes in multiple antibiotic resistant Salmonella typhimurium DT104.

Antibiotic resistance in pathogenic bacteria is a problem in both industrialized and developing countries. This is especially evident in Salmonella typhimurium, a foodborne pathogen that causes gastrointestinal and systemic disease throughout the world. S. typhimurium DT104 further poses a major health concern due to its apparent enhanced ability to acquire multiple antibiotic resistance genes and its putative hypervirulent phenotype. Recently, we demonstrated that multiresistant S. typhimurium do not appear to be more invasive than non-resistant cohorts. In the present study, we evaluated the presence of Salmonella pathogenicity island 1 (SPI1) flanking and internal sequences in over 400 isolates of multiresistant S. typhimurium. With these same isolates, we also used a tissue culture invasion assay to evaluate a potential relationship between antibiotic exposure and a hyperinvasive phenotype. Our studies revealed that SPI1 flanking sequences are similar in multiresistant and non-resistant S. typhimurium. Furthermore, we failed to identify any isolates that were hyperinvasive in the presence of any of the 14 antibiotics evaluated. These results further indicate that the putative hypervirulence of multiresistant S. typhimurium is not likely to occur at the level of invasion.

Anti-Bacterial Agents↗
PubMed2000

Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture.

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in </=4 h after enrichment.

Animals↗
PubMed1999

Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.

Drug Resistance, Microbial↗
PubMed1998

Use of the alveolar-arterial oxygen gradient in the assessment of acute pulmonary embolism.

To evaluate the utility of the alveolar-arterial (A-a) oxygen gradient in the diagnosis of acute pulmonary embolism (PE), a retrospective analysis was done of consecutive emergency department patients who underwent pulmonary angiography for the presumed diagnosis of acute PE. Patients were categorized into two groups depending on the presence or absence of prior cardiopulmonary disease. Arterial blood gas samples were chosen for analysis only if obtained when the PE first was suspected clinically (before lung scans or angiograms) and the patient was breathing room air. A total of 152 patients met all study criteria; 59 patients (39%) had angiographically documented emboli. In comparison with the study patients in whom PE was excluded, there was no significant difference in mean PaO2 (64 v 67 mm Hg) or A-a gradient (39 v 36 mm Hg). Various combinations of the A-a gradient and blood gas levels failed to exclude PE in more than 35% of patients with no prior cardiovascular disease and in 25% of patients with prior cardiovascular disease. The A-a gradient did show a linear correlation with the severity of the PE, as assessed by the PaO2 (r = -0.87) and pulmonary artery mean pressure (r = 0.63). These results indicate that the A-a oxygen gradient, in combination with blood gas levels, may contribute to the formulation of a clinical assessment. However, these laboratory parameters are of insufficient discriminant value to permit exclusion of the diagnosis of PE.

Acute Disease↗
PubMed1998

Mutation of a putative amphipathic alpha-helix in the third intracellular domain of the platelet-activating factor receptor disrupts receptor/G protein coupling and signaling.

Platelet-activating factor (PAF) is a potent phospholipid mediator that interacts with G protein-coupled PAF receptors to elicit diverse physiological and pathophysiological actions. We recently demonstrated that the third intracellular domain of the rat PAF receptor (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C-activating G proteins. Here, we report identification of a putative amphipathic helix in the third intracellular domain of the rPAFR and the effects of mutational disruption of its amphipathic character on G protein coupling of and signaling by the rPAFR. Modeling of the third intracellular domain and adjacent transmembrane regions of the rPAFR identified a single amphipathic helix located in the amino-terminal region of the third intracellular domain of the receptor. Baby hamster kidney cells were transiently transfected with cDNAs encoding the rPAFR or rPAFR mutants in which nonconserved substitutions were made separately in the hydrophobic or polar face of this amphipathic helix. The number and affinity of binding sites for specific PAF receptor antagonist WEB2086 were identical in membranes prepared from rPAFR and amphipathic helix mutant PAFR transfectants. However, only membranes derived from rPAFR transfectants possessed high affinity PAF binding sites that were sensitive to the G protein-uncoupling effects of guanosine-5'-O-(3-thio)triphosphate. These results show that substitutions into either face of the amphipathic helical domain abolished the ability of the rPAFR to undergo coupling to G proteins to form a high affinity agonist/receptor/G protein ternary complex. To examine the effects of these mutations on rPAFR signaling, PAF-stimulated inositol phosphate accumulation was determined in cells transfected with cDNAs encoding the wild-type or amphipathic helix mutant PAFRs. Although PAF stimulated 10-fold increases in inositol phosphate accumulation in rPAFR transfectants, it had no effects on inositol phosphate accumulation in amphipathic helix mutant PAFR transfectants. These results suggest that an amphipathic helix located in the amino-terminal region of the third intracellular domain of the rPAFR is required for its coupling to and activation of G proteins. This study provides the first insight into the structure of the receptor interface for G protein coupling of a PAFR and suggests a conserved role of amphipathic helices in G protein coupling of receptors ranging from those for biogenic amines to the phospholipid mediator PAF.

Amino Acid Sequence↗
PubMed1998

Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins.

Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species. These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.

Amino Acid Sequence↗
PubMed1997

Assessing bedside cardiologic examination skills using "Harvey," a cardiology patient simulator.

OBJECTIVE: To assess the cardiovascular physical examination skills of emergency medicine (EM) housestaff and attending physicians. METHODS: Prospective, cohort assessment of EM housestaff and faculty performance on 3 valvular abnormality simulations (mitral regurgitation, mitral stenosis, and aortic regurgitation) conducted on the cardiology patient simulator, "Harvey." Participants examined each of the 3 study disease simulations and proposed a diagnosis (session I). They were then given a cardiac examination form and repeated the programmed simulations (session II). The examination form was used to prompt physicians to interpret 23 separate cardiac findings for each simulation in a multiple-choice format. RESULTS: Forty-six EM housestaff (PGY1-3) and attending physicians were tested over a 2-month study period. Physician responses did not differ significantly among the different levels of postgraduate training. The overall correct response rates for participants were 59% for aortic regurgitation, 48% for mitral regurgitation, and 17% for mitral stenosis. For aortic regurgitation, recognition of a widened pulse pressure and recognition of diastolic decrescendo murmur were associated with a correct diagnosis (p < 0.01). For mitral regurgitation, correct assessment of the contour of the holosystolic murmur predicted a correct diagnosis (p < 0.001). For mitral stenosis, proper characterization of the mitral area diastolic murmur predicted a correct diagnosis (p < 0.001). CONCLUSION: Housestaff and faculty had difficulty establishing a correct diagnosis for simulations of 3 common valvular heart diseases. However, accurate recognition of a few critical signs was associated with a correct diagnosis in each simulation. Training programs may need to focus attention on selected key components of the cardiovascular examination to facilitate teaching of physical diagnosis.

Clinical Competence↗
PubMed1996

The third intracellular domain of the platelet-activating factor receptor is a critical determinant in receptor coupling to phosphoinositide phospholipase C-activating G proteins. Studies using intracellular domain minigenes and receptor chimeras.

Platelet activating factor (PAF) is a potent phospholipid mediator which elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads to activation of G protein(s) that stimulate phosphoinositide phospholipase C and subsequent intracellular signaling responses. To identify the potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) production mediated by the co-transfected rPAFR cDNA. Although transfection of the rPAFR1i and rPAFR2i minigenes had no effects on PAF-stimulated signaling, transfection of the rPAFR3i minigene inhibited PAF-stimulated IP production by approximately 50% compared to controls. The rPAFR3i domain did not inhibit IP production mediated by the multifunctional rat pituitary adenylate cyclase-activating polypeptide receptor (rPACAPR), demonstrating the specificity of the competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunctional transmembrane variant of the rPACAPR (rPACAPR2) that activates only adenylyl cyclase. The rPACAPR2/rPAFR3i chimera responded to PACAP with increases in IP production which were attenuated nearly completely in cells cotransfected with the rPAFR3i domain. In contrast, PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. These results suggest that the third intracellular loop of the rPAFR is a primary determinant in its coupling to phosphoinositide phospholipase C-activating G proteins, providing the first insight into the molecular basis of interaction of PAFRs with signal-transducing G proteins.

Amino Acid Sequence↗