Iron-dependent reconfiguration of the proteome underlies the intracellular lifestyle of Brucella abortus.

Brucella ssp. is a facultative intracellular pathogen that causes brucellosis, a worldwide zoonosis that affects a wide range of mammals including humans. A critical step for the establishment of a successful Brucella infection is its ability to survive within macrophages. To further understand the mechanisms that Brucella utilizes to adapt to an intracellular lifestyle, a differential proteomic study was performed for the identification of intracellular modulated proteins. Our results demonstrated that at 48 hours post-infection Brucella adjusts its metabolism in order to survive intracellularly by modulating central carbon metabolism. Remarkably, low iron concentration is likely the dominant trigger for reprogramming the protein expression profile. Up-regulation of proteins dedicated to reduce the concentration of reactive oxygen species, protein chaperones that prevent misfolding of proteins, and proteases that degrade toxic protein aggregates, suggest that Brucella protects itself from damage likely due to oxidative burst. This proteomic analysis of B. abortus provides novel insights into the mechanisms utilized by Brucella to establish an intracellular persistent infection and will aid in the development of new control strategies and novel targets for antimicrobial therapy.

Antibiotic susceptibility of isolates of Bacillus anthracis, a bacterial pathogen with the potential to be used in biowarfare.

Bacillus anthracis is a bacterial species that could be used in a bioterrorist attack. We tested a collection of isolates with a range of relevant antimicrobial compounds. All isolates tested were susceptible to ciprofloxacin and doxycycline. Penicillin and amoxicillin, with or without clavulanate, showed in vitro activity against all B. anthracis isolates. Ceftriaxone demonstrated lower-level in vitro activity compared to penicillin-related compounds against B. anthracis. In vitro data from this study are in keeping with available guidelines.

Characterization of the caprine model for ruminant brucellosis.

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.

Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis.

The potential use of Bacillus anthracis as a weapon of mass destruction poses a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay. We have developed a real-time PCR-based assay for the specific detection of B. anthracis by taking advantage of the unique nucleotide sequence of the B. anthracis rpoB gene. Variable region 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 B. anthracis strains and 20 other related bacilli, and four nucleotides specific for B. anthracis were identified. PCR primers were selected so that two B. anthracis-specific nucleotides were at their 3' ends, whereas the remaining bases were specific to the probe region. This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET). The assay was found to be specific for 144 B. anthracis strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception of one strain. The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h. Therefore, the rpoB-FRET assay could be used as a new chromosomal marker for rapid detection of B. anthracis.

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.

A PCR assay for identification of Enterococcus faecium.

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The prevalence and severity of enterococcal infections, the mortality rate from such infections, and the antibiotic resistance of enterococci are often species dependent. Since conventional biochemical methods fail to differentiate E. faecium from certain newly described enterococcal species, a PCR-based assay was developed for the rapid identification of E. faecium.

Development of a diagnostic polymerase chain reaction assay for detection of Mycoplasma hominis.

A polymerase chain reaction (PCR)-based assay was developed for the detection of Mycoplasma hominis. This assay generates a 152-bp PCR product which was part of an initial 471-bp M. hominis genomic DNA fragment. The 471-bp DNA fragment was shown by hybridization analysis to be unique for M. hominis. The PCR assay can amplify as few as 18 molecules of target DNA. This diagnostic assay offers potential for wide clinical application as it is rapid and can be successfully performed on crude sample preparations from a variety of media or biopsies. The use of this assay should aid in defining the aetiologic and pathologic roles played by M. hominis and thereby benefit patients.

Development of a DNA probe for Ureaplasma urealyticum.

Ureaplasma urealyticum has been associated with a variety of disease conditions in humans. However, its exact etiologic role has not been well established because of the difficulties encountered in cultural diagnosis and the time needed for positive identifications. A DNA probe which is specific for a target DNA sequence unique to this suspected pathogen offers a rapid, sensitive and specific means of diagnosis. This study details the development of a polymerase chain reaction system for U. urealyticum. Using conventional hybridization techniques, a cloned genomic fragment was found to be specific for this organism. Sequencing of part of this probe DNA permitted the assignment of oligonucleotide primers which amplified a 186 bp target segment. This PCR system is specific for U. urealyticum but not for other closely related species of mycoplasma. This highly sensitive diagnostic technique will aid in determining the etiologic role, tissue tropism and dynamics of pathogenesis of this organism, and thereby result in better patient care.