RESUMO
From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.
Assuntos
Brucella/isolamento & purificação , Brucelose/veterinária , Raposas , Linfonodos/microbiologia , Animais , Áustria , Proteínas da Membrana Bacteriana Externa/genética , Brucella/classificação , Brucella/genética , Brucelose/epidemiologia , Brucelose/microbiologia , República Tcheca/epidemiologia , Genótipo , Geografia , Mandíbula , Ochrobactrum anthropi/classificação , Ochrobactrum anthropi/isolamento & purificação , RNA Ribossômico 16S/genética , Recombinases Rec A/genética , Análise de SequênciaRESUMO
Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. are phenotypically and genetically closely related pathogens that may cause disease with similar clinical presentation. Consequently, difficulties in their identification and differentiation have been reported. In this study, a sensitive recA gene-based multi-primer single-target PCR (MP-ST-PCR) was developed that allowed the specific detection and differentiation of these clinically relevant pathogens. The specificity of the assay was evaluated using a representative panel of 50 O. anthropi and 16 O. intermedium strains and the type strains of all Brucella spp. Detection limits for purified DNA from O. anthropi, O. intermedium and Brucella melitensis were 100, 10 and 100 fg, respectively. Brucella DNA was also successfully detected in various clinical specimens from a human patient with culture-proven brucellosis and from a Brucella-infected sheep and its aborted fetuses. The sensitivity of the MP-ST-PCR was comparable to that of an evaluated in-house Brucella real-time PCR assay. The developed assay closes a diagnostic gap and provides a simple but robust tool for the sensitive detection and correct identification of O. anthropi, O. intermedium and Brucella spp.
Assuntos
Técnicas de Tipagem Bacteriana , Brucella/isolamento & purificação , DNA Bacteriano/análise , Infecções por Bactérias Gram-Negativas/microbiologia , Ochrobactrum/isolamento & purificação , Recombinases Rec A/genética , Animais , Brucella/genética , Brucelose/diagnóstico , Primers do DNA , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , Ochrobactrum/genética , Ochrobactrum anthropi/genética , Ochrobactrum anthropi/isolamento & purificação , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , OvinosRESUMO
Based on the recently completed genomic sequence of Chromobacterium violaceum American Type Culture Collection (ATCC) 12472 a multiplex PCR assay targeting the prgI, spaO, invG, and sipB genes of the Salmonella SPI-1 homologue type-III secretion system was developed. PCR products of 255bp (prgI), 749bp (spaO), 1685bp (invG), and 1752bp (sipB) were successfully amplified simultaneously in a single reaction with all Chr. violaceum strains investigated whereas other bacteria tested negative. The detection limit for pure cultures in multiplex PCR analysis was 100CFU. The developed assay significantly improves rapid identification of Chr. violaceum and allows its differentiation from closely related organisms.
Assuntos
Proteínas de Bactérias/genética , Chromobacterium/genética , Reação em Cadeia da Polimerase/métodos , Animais , Proteínas de Bactérias/química , Chromobacterium/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genéticaRESUMO
Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.
