Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, accurate method for identifying bacteria and fungi recovered on agar culture media. We report herein a method for the direct identification of bacteria in positive blood culture broths by MALDI-TOF mass spectrometry. A total of 212 positive cultures were examined, representing 32 genera and 60 species or groups. The identification of bacterial isolates by MALDI-TOF mass spectrometry was compared with biochemical testing, and discrepancies were resolved by gene sequencing. No identification (spectral score of <1.7) was obtained for 42 (19.8%) of the isolates, due most commonly to insufficient numbers of bacteria in the blood culture broth. Of the bacteria with a spectral score of >1.7, 162 (95.3%) of 170 isolates were correctly identified. All 8 isolates of Streptococcus mitis were misidentified as being Streptococcus pneumoniae isolates. This method provides a rapid, accurate, definitive identification of bacteria within 1 h of detection in positive blood cultures with the caveat that the identification of S. pneumoniae would have to be confirmed by an alternative test.In the early 1970s the first semiautomated blood culture system, the radiometric Bactec system, was introduced into the clinical microbiology laboratory. In subsequent years this system was refined with the development of fully automated, closed, continuously monitoring systems for the detection of microbial growth. Recently, a commercial real-time PCR system (LightCycler SeptiFast; Roche Molecular Systems) was introduced with the hope that culture-based systems could be replaced with this technology; however, initial reports documented that this system can be used as a complement to but not a replacement for the current generation of automated systems (16,17,19). Because culture-based systems will be used in the near future, accurate, rapid identification methods are still needed. As with blood culture systems, a transition in identification systems began in the early 1970s with the introduction of commercial biochemical strips and panels and then with the rapid development and refinement of automated instruments that inoculate, incubate, interpret, and report microbial identifications. Currently, most bacteria and fungi can be identified with these systems in a few hours to 1 to 2 days, with slow-growing or metabolically inert organisms requiring additional time or supplementary tests. The identification of organisms recovered in blood culture broths requires an initial subculture of the broth and overnight incubation to obtain isolated colonies for further testing or the concentration of the bacteria or fungi by centrifugation before further processing. In general, the approach of concentrating organisms by centrifugation and then identification by rapid biochemical tests (1a), fluorescent in situ hybridization (FISH) (4, 6, 15, 18), or commercial biochemical systems is accurate, although the limitations of incubation delays, the need for supple...

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The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and is required for the production of an unknown quorum-sensing molecule. In a screen to identify rhomboid-encoding genes from Proteus mirabilis, tatA was identified as a multicopy suppressor and restored extracellular signal production as well as complementing all other phenotypes of a Prov. stuartii aarA mutant. TatA is a component of the twin-arginine translocase (Tat) protein secretion pathway and likely forms a secretion pore. By contrast, the native tatA gene of Prov. stuartii in multicopy did not suppress an aarA mutation. We find that TatA in Prov. stuartii has a short N-terminal extension that was atypical of TatA proteins from most other bacteria. This extension was proteolytically removed by AarA both in vivo and in vitro. A Prov. stuartii TatA protein missing the first 7 aa restored the ability to rescue the aarA-dependent phenotypes. To verify that loss of the Tat system was responsible for the various phenotypes exhibited by an aarA mutant, a tatC-null allele was constructed. The tatC mutant exhibited the same phenotypes as an aarA mutant and was epistatic to aarA. These data provide a molecular explanation for the requirement of AarA in quorum-sensing and uncover a function for the Tat protein export system in the production of secreted signaling molecules. Finally, TatA represents a validated natural substrate for a prokaryotic rhomboid protease.

In this study, we identified a transposon insertion in a novel gene, designated disA, that restored swarming motility to a putrescine-deficient speA mutant of Proteus mirabilis. A null allele in disA also increased swarming in a wild-type background. The DisA gene product was homologous to amino acid decarboxylases, and its role in regulating swarming was investigated by examining the expression of genes in the flagellar cascade. In a disA mutant background, we observed a 1.4-fold increase in the expression of flhDC, which encodes FlhD 2 C 2 , the master regulator of the flagellar gene cascade. However, the expressions of class 2 (fliA, flgM) and class 3 (flaA) genes were at least 16-fold higher in the disA background during swarmer cell differentiation. Overexpression of DisA on a high-copy-number plasmid did not significantly decrease flhDC mRNA accumulation but resulted in a complete block in mRNA accumulation for both fliA and flaA. DisA overexpression also blocked swarmer cell differentiation. The disA gene was regulated during the swarming cycle, and a single-copy disA::lacZ fusion exhibited a threefold increase in expression in swarmer cells. Given that DisA was similar to amino acid decarboxylases, a panel of decarboxylated amino acids was tested for effects similar to DisA overexpression, and phenethylamine, the product of phenylalanine decarboxylation, was capable of inhibiting both swarming and the expression of class 2 and class 3 genes in the flagellar regulon. A DisA-dependent decarboxylated amino acid may inhibit the formation of active FlhD 2 C 2 heterotetramers or inhibit FlhD 2 C 2 binding to DNA.Proteus mirabilis is capable of a complex differentiation that results in the formation of an elongated swarmer cell from a short rod. This differentiation occurs upon the transfer of cells from a liquid medium to a solid surface and is marked by elongated, aseptate, and hyperflagellated cells. Reviews on this process provide additional details (12,15,21,22,23). Swarmer cells align themselves in multicellular rafts and move in a coordinated fashion to a new location (16). The swarmer cell represents a transient state, and after a period of migration, the swarmer cell dedifferentiates back to the vegetative cell in a process termed consolidation. After a new period of growth, the consolidated vegetative cells then differentiate back to swarmer cells, followed by a new period of cell migration. This cyclic process results in the formation of swarming rings on an agar plate, where each ring represents one cycle of differentiation/dedifferentiation.A large number of genes that function in diverse aspects of physiology and metabolism are required for swarming and have revealed the complexity of this process (see references 3-6, 11, 13, 14, 18, 26, and 27; reviewed in reference 22). Swarmer cell differentiation is dependent on certain environmental conditions that include a solid surface, inhibition of flagellar rotation, and cell-cell signaling (1, 6, 27).In P. mirabilis, a key regulator of both flagellin e...

The accurate identification of Leishmania species is important for the treatment of infected patients. Molecular methods offer an alternative to time consuming traditional laboratory techniques for species determination. We redesigned a 7SL rRNA gene based PCR and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including: