Previous studies have shown that a 54 -S cascade regulates the expression of a few key lipoproteins in Borrelia burgdorferi, the agent of Lyme disease. Here, we demonstrate that these sigma factors, both together and independently, regulate a much more extensive number of genes and cellular processes. Microarray analyses of 54 and S mutant strains identified 305 genes regulated by 54 and 145 regulated by S , whereas the 54 -S regulatory cascade appears to control 48 genes in B. burgdorferi. In silico analyses revealed that nearly 80% of genes with altered expression in the 54 mutant were linked to potential 54 -dependent promoters. Many 54 -regulated genes are expressed in vivo, and through genetic complementation of the mutant, we demonstrated that 54 was required by B. burgdorferi to infect mammals. Surprisingly, 54 mutants were able to infect Ixodes scapularis ticks and be maintained for at least 24 wk after infection, suggesting the 54 -S regulatory network was not involved in long-term survival in ticks. However, 54 mutants did not enter the salivary glands during tick feeding, indicating that 54 -regulated genes were involved in the transmission process.infectivity ͉ microarray ͉ Lyme ͉ transcription

Despite extensive study, little is known about the origins of the mutualistic bacterial endosymbionts that inhabit approximately 10% of the world's insects. In this study, we characterized a novel opportunistic human pathogen, designated “strain HS,” and found that it is a close relative of the insect endosymbiont Sodalis glossinidius. Our results indicate that ancestral relatives of strain HS have served as progenitors for the independent descent of Sodalis-allied endosymbionts found in several insect hosts. Comparative analyses indicate that the gene inventories of the insect endosymbionts were independently derived from a common ancestral template through a combination of irreversible degenerative changes. Our results provide compelling support for the notion that mutualists evolve from pathogenic progenitors. They also elucidate the role of degenerative evolutionary processes in shaping the gene inventories of symbiotic bacteria at a very early stage in these mutualistic associations.

We used microarrays and real-time reverse transcription-PCR to analyze the global transcriptional response of Mycobacterium tuberculosis to low pH in vitro, which may mimic an environmental signal encountered by phagocytosed mycobacteria. Eighty-one genes were differentially expressed >1.5-fold, including many involved in fatty acid metabolism. The most highly induced genes showed homology with nonribosomal peptide synthetases/polyketide synthases.Among the first steps in human infection with Mycobacterium tuberculosis is phagocytosis of the bacteria by macrophages in the lung. The bacilli can survive and multiply in this normally hostile environment because the phagosomes do not fully mature into phagolysosomes (4,8,48,52). Phagosomes containing mycobacteria begin to acidify rapidly after phagocytosis. If the bacilli are viable, the pH drops below 6 and then may rise over several hours to approximately 6.5. Dead mycobacteria, however, do not block phagosomal acidification, and the pH drops rapidly to 5.5 or lower (21,37,49). These data suggest active inhibition of phagosomal acidification by the bacilli. Acidification itself may be a signal used by mycobacteria to induce the expression of genes needed to alter phagosomal maturation.Whole-genome microarrays have been used previously to determine gene expression patterns of tubercle bacilli in response to several different environmental conditions (34,42,54). One key to the effective use of microarrays for expression studies is to have a well-controlled experimental protocol in which RNA can be rapidly extracted from uniform populations of cells. In our investigation of changes in gene expression following phagocytosis, we chose to use an in vitro acid shock instead of isolating phagocytosed mycobacteria to avoid complications due to difficulties in rapidly isolating mycobacterial RNA from a mixture of mycobacteria and macrophages and in synchronizing phagocytosis and obtaining a uniform population of mycobacteria.Microarray analysis of transcription after acid shock. M. tuberculosis H37Rv (TMC102) bacteria were grown in Middlebrook 7H9 broth (Difco, Detroit, Mich.) supplemented with 10% (vol/vol) albumin-dextrose-catalase (Difco) and 0.05% (vol/vol) Tween 80 (Sigma, St. Louis, Mo.) at 37°C to an A 600 of Х0.5 on a rotating platform (50 rpm). Bacteria were harvested by centrifugation (5 min, 6,000 ϫ g) at room temperature, resuspended in fresh prewarmed 7H9-T (pH 6.9) medium, and allowed to recover for 3 h at 37°C with shaking. Cells were harvested by centrifugation (1 min, 25,000 ϫ g, 37°C), resuspended in either prewarmed 7H9-T (pH 6.9) or acidic 7H9-T (pH 5.5) medium, and incubated at 37°C with shaking. At 15 and 30 min, samples of each culture were removed and RNA was extracted by a modification of the method of DesJardin (10). The overnight acid shock was performed essentially as described above, except that the cells were incubated for 18 h in the normal or acid broth and the starting cell density was A 600 Х 0.28 to ensure that the final cell density would...

Among US veterans, ST131, primarily its H30 subclone, accounts for most antimicrobial-resistant E. coli and is the dominant E. coli strain overall. Possible contributors include multidrug resistance, extensive virulence gene content, and ongoing transmission. Focused attention to ST131, especially its H30 subclone, could reduce infection-related morbidity, mortality, and costs among veterans.

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Inc., Salt Lake City, UT) Blood Culture (BC) panel can identify > 25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 hour. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 of 92 pathogens (91%) covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven MRSA and VRE. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.

The genus Nocardia has undergone rapid taxonomic expansion in recent years, and an increasing number of species are recognized as human pathogens. Many established species have predictable antimicrobial susceptibility profiles, but sufficient information is often not available for recently described organisms. Additionally, the effectiveness of sulfonamides as first-line drugs for Nocardia has recently been questioned. This led us to review antimicrobial susceptibility patterns for a large number of molecularly identified clinical isolates. Susceptibility results were available for 1,299 isolates representing 39 different species or complexes, including 11 that were newly described, during a 6-year study period. All tested isolates were susceptible to linezolid. Resistance to trimethoprim-sulfamethoxazole (TMP-SMX) was rare (2%) except among Nocardia pseudobrasiliensis (31%) strains and strains of the N. transvalensis complex (19%). Imipenem susceptibility varied for N. cyriacigeorgica and N. farcinica, as did ceftriaxone susceptibility of the N. nova complex. Resistance to more than one of the most commonly used drugs (amikacin, ceftriaxone, TMP-SMX, and imipenem) was highest for N. pseudobrasiliensis (100%), N. transvalensis complex (83%), N. farcinica (68%), N. puris (57%), N. brasiliensis (51%), N. aobensis (50%), and N. amikacinitolerans (43%). Thus, while antimicrobial resistance can often be predicted, susceptibility testing should still be considered when combination therapy is warranted, for less well characterized species or those with variable susceptibility profiles, and for patients with TMP-SMX intolerance.

bShigella species are so closely related to Escherichia coli that routine matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) cannot reliably differentiate them. Biochemical and serological methods are typically used to distinguish these species; however, "inactive" isolates of E. coli are biochemically very similar to Shigella species and thus pose a greater diagnostic challenge. We used ClinProTools (Bruker Daltonics) software to discover MALDI-TOF MS biomarker peaks and to generate classification models based on the genetic algorithm to differentiate between Shigella species and E. coli. Sixtysix Shigella spp. and 72 E. coli isolates were used to generate and test classification models, and the optimal models contained 15 biomarker peaks for genus-level classification and 12 peaks for species-level classification. We were able to identify 90% of E. coli and Shigella clinical isolates correctly to the species level. Only 3% of tested isolates were misidentified. This novel MALDI-TOF MS approach allows laboratories to streamline the identification of E. coli and Shigella species.

b Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species-and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications. Recent decades have seen advances in automation of traditional phenotypic and biochemical methods for microbial identification (ID), and advances in sequencing and the proliferation of genomic data hold great promise for further improvements. The development of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has brought microbial diagnostics to another cusp of rapid development. The speed and low cost of bacterial identification by MALDI-TOF MS make it an attractive technology in the clinical microbiology laboratory, and it has shown promise for identification of Gram-positive cocci (2, 6, 8), enteric and nonfermenting Gram-negative rods (11,21,24), HACEK organisms (10), anaerobes (14,17,19,20,31), and broad cohorts of clinically relevant bacteria (3,4,22,27,30).Commercial MALDI-TOF systems identify a broad range of microorganisms based on analysis of unique "fingerprints" of abundant proteins from whole cells or cellular extracts (15,23,26,28). These profiles are searched against databases of reference spectra, and similarity scores for the top database matches are used to determine the identification of unknown isolates. As observed previously, a systematic evaluation of scoring criteria on diverse isolates could improve results (2,10,25,27,29). Identification may be complicated when multiple species-or genus-level matches are among the top 10 results. Most current publications on the MALDI Biotyper system (Bruker Daltonics, Billerica, MA) do not address these complicated situations; however, one example where this problem is addressed is the use of the "10% rule," which stat...