ABSTRACT
High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.
Subject(s)
DNA Fingerprinting/methods , Staphylococcus/classification , Staphylococcus/genetics , Base Sequence , DNA, Bacterial/chemistry , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Heteroduplex Analysis , Molecular Sequence Data , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Sensitivity and Specificity , Sequence Alignment , Sequence Analysis, DNA , Species Specificity , Transition TemperatureABSTRACT
Cardiobacterium valvarum, a fastidious Gram-negative bacterium, was detected in the aortic valve of a previously healthy 63-year-old man by broad-range PCR and 16S rRNA gene sequencing. In contrast to the patients in five previously published cases, our patient had neither a congenital bicuspid nor a prosthetic aortic valve. Here, we present a case of C. valvarum native tricuspid aortic valve infective endocarditis and a review of the literature.
Subject(s)
Aortic Valve/pathology , Cardiobacterium/isolation & purification , Endocarditis, Bacterial/microbiology , Heart Valve Diseases/microbiology , Polymerase Chain Reaction , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/therapeutic use , Aortic Valve/microbiology , Cefotaxime/administration & dosage , Cefotaxime/therapeutic use , Endocarditis, Bacterial/drug therapy , Endocarditis, Bacterial/surgery , Gentamicins/administration & dosage , Gentamicins/therapeutic use , Heart Valve Diseases/pathology , Humans , Male , Middle AgedABSTRACT
A rare case of a severe prosthetic joint infection in a 71-year-old immunocompetent woman is presented. Listeria monocytogenes was identified in two consecutive samples using broad-range PCR and sequencing, whereas cultivation remained negative for the first sample and streptococci of a non-group A streptococci, non-group B streptococci type were detected for the second one. This report demonstrates that the phenotypic approach may lead to misidentification of L. monocytogenes in a routine clinical setting. Molecular methods of pathogen detection might be useful when a rare and/or unexpected micro-organism is present or the sample is collected during antibiotic treatment.
Subject(s)
Knee Prosthesis/microbiology , Listeria monocytogenes/classification , Listeria monocytogenes/isolation & purification , Listeriosis/diagnosis , Prosthesis-Related Infections/diagnosis , Aged , Culture Media , DNA, Bacterial/analysis , Female , Genotype , Humans , Listeria monocytogenes/genetics , Listeria monocytogenes/physiology , Listeriosis/microbiology , Phenotype , Polymerase Chain Reaction , Prosthesis-Related Infections/microbiology , Sequence Analysis, DNAABSTRACT
A case of blood culture-negative aortic and mitral valve infective endocarditis caused by the bacterium Tropheryma whipplei is reported. A 33-year-old man showed no clinical manifestations of Whipple's disease, with the exception of arthralgia as given in his history. The disease was complicated by recurrent cerebral and left upper limb embolization of vegetations. The causative agent was detected in a valve sample using broad-range polymerase chain reaction. The patient underwent successful surgery (aortic and mitral valve replacement) and his clinical condition during six-month antibiotic therapy was good.
Subject(s)
Actinomycetales Infections/diagnosis , Aortic Valve , Endocarditis, Bacterial/diagnosis , Heart Valve Diseases/diagnosis , Mitral Valve , Tropheryma , Whipple Disease/diagnosis , Adult , Humans , MaleABSTRACT
The authors present a case report of a patient with sepsis caused by Gram negative rod Capnocytophaga canimorsus resulting from a dog bite. The infection had a course of septic shock progressing into multiorgan failure and serious ischemic damage to the extremities. The etiologic agent was identified utilizing molecular genetic methods and its detailed microbiologic characteristics are provided below. The report also outlines diagnostic and therapeutic options of this otherwise most likely under-diagnosed infection.
