Effect of delayed entry on performance of the BACT/ALERT FAN PLUS bottles in the BACT/ALERT VIRTUO blood culture system.

Delayed entry of patient blood culture samples into a microbial detection system is unavoidable at times, due to off-shift staffing or transporting samples to centralized laboratories. Pre-incubation time and temperature of blood culture bottles are the most critical factors impacting recovery and detection of microorganisms. A total of 1377 BACT/ALERT® (BTA) Fastidious Antimicrobial Neutralization (FAN® PLUS) bottles (FA PLUS, FN PLUS, and PF PLUS) were tested after delayed entry times of 24 and 36 h at 20-25 °C (room temperature, RT) prior to loading into the BACT/ALERT® VIRTUO® microbial detection system (VIRTUO). Clinically relevant organisms were inoculated into bottles with 5-84 colony forming units (CFU) per bottle, and human blood (0 to 10 mL), and then loaded into the VIRTUO. When bottles were loaded without delay, a mean time to detection (TTD) of 9.6 h was observed. For delayed bottles, the TTD reported by the VIRTUO was added to the 24-h and 36-h delay times and resulted in average time to results of 32.5 h and 42.5 h, respectively. The FAN PLUS bottles in conjunction with the VIRTUO produced acceptable results when delays up to 24 h at 20-25 °C occur in loading.

Performance of Vitek MS v3.0 for Identification of Mycobacterium Species from Patient Samples by Use of Automated Liquid Medium Systems.

The accuracy and robustness of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system was evaluated by identifying mycobacteria from automated liquid-medium systems using patient samples. This is the first report to demonstrate that proteins within the liquid medium, its supplements, and decontamination reagents for nonsterile patient samples do not generate misidentification or false-positive results by use of the Vitek MS v3.0 system. Prior to testing with patient samples, a seeded-culture study was conducted to challenge the accuracy of the Vitek MS system at identifying mycobacteria from liquid medium by mimicking a clinical workflow. Seventy-seven Mycobacterium strains representing 21 species, seeded in simulated sputum, were decontaminated, inoculated into BacT/Alert MP liquid culture medium, incubated until positivity, and identified using the Vitek MS system. A total of 383 liquid cultures were tested, of which 379 (99%) were identified correctly to the species/complex/group level, 4 (1%) gave a "no-identification" result, and no misidentifications were observed. Following the simulated-sputum study, a total of 73 smear-positive liquid-medium cultures detected using BD BBL MGIT and VersaTREK Myco liquid media were identified by the Vitek MS system. Sixty-four cultures (87.7%) were correctly identified to the species/complex/group level; 7 (9.6%) resulted in no identification; and 2 (2.7%) were misidentified at the species level. These results indicate that the Vitek MS v3.0 system is an accurate tool for routine diagnostics of Mycobacterium species isolated from liquid cultures.

Progress in proteomics for clinical microbiology: MALDI-TOF MS for microbial species identification and more.

Although classical proteomic approaches are still used regularly in routine clinical diagnostic procedures, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) MS has recently moved into diagnostic microbiology laboratories. MALDI-TOF MS is currently replacing phenotypic microbial identification. Many laboratories now use MALDI-TOF MS for its high efficiency, both from a diagnostic and a cost-per-analysis point of view. The US FDA has now cleared two of the commercially available systems for in vitro diagnostics. This will further spark development of MS applications in antimicrobial susceptibility testing and epidemiology. This review summarizes the state of affairs of MALDI-TOF MS in clinical microbiology; however, this is an active field of research subject to rapid evolution. We emphasize assessment of the clinical relevance and studies focusing on data obtained through comparative analyses of different MALDI-TOF MS instrumentation and multicenter validation studies. The future of MALDI-TOF MS, including antimicrobial susceptibility testing and epidemiological typing, is also highlighted.

Rapid inactivation of Mycobacterium and nocardia species before identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained.

A novel liquid media mycobacteria extraction method for MALDI-TOF MS identification using VITEK® MS.

A low-cost identification method that can be performed directly from a positive liquid medium culture is needed for the diagnosis of mycobacterial infections. Here, we describe a novel, cost-effective, and validated method that allows for direct and rapid identification of mycobacteria from a positive liquid culture using VITEK® MS with a total process duration under 45min. From a liquid mycobacteria culture a 3.0mL aliquot is removed 24-72h post positivity and centrifuged to create a pellet. After decanting, the tube is blotted dry, the pellet is re-suspended in 0.5mL of 70% ethanol and then transferred into a 2.0mL tube containing glass beads. Mycobacteria are disrupted mechanically followed by a 10min. incubation at room temperature to complete inactivation. Inactivated material is pelleted by centrifugation and then re-suspended in 10µL of 70% formic acid and 10µL of acetonitrile. After centrifugation, 1µL of supernatant (protein extract) is deposited onto target slide, allowed to dry, and then 1µL CHCA matrix is added. A seeded study was conducted to demonstrate the reliability of the method, a total of 251 culture samples obtained from automated culture systems (BacT/ALERT® MP bottles, BACTEC MGIT™ 960 tubes, and VersaTREK® Myco bottles), were tested and resulted in 98.8% correct identification. Reproducibility was shown by testing three organisms across three reagent lots, between four laboratory technicians, over the course of five days for three liquid media systems resulting in a total of 180 deposits with an overall correct identification of 98.9% with the remaining results giving no identification. Additional studies were performed including comparison of different mechanical disruption techniques, stability of frozen extracts, and stability of slide deposits to allow for flexibility in a routine clinical workflow. The described method proved to be safe while providing consistent and reproducible results for different species of mycobacteria and is compatible with the three most widely used liquid media medium detection systems.

A MALDI-TOF MS database with broad genus coverage for species-level identification of Brucella.

Brucella are highly infectious bacterial pathogens responsible for a severely debilitating zoonosis called brucellosis. Half of the human population worldwide is considered to live at risk of exposure, mostly in the poorest rural areas of the world. Prompt diagnosis of brucellosis is essential to prevent complications and to control epidemiology outbreaks, but identification of Brucella isolates may be hampered by the lack of rapid and cost-effective methods. Nowadays, many clinical microbiology laboratories use Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF MS) for routine identification. However, lack of reference spectra in the currently commercialized databases does not allow the identification of Brucella isolates. In this work, we constructed a Brucella MALDI-TOF MS reference database using VITEK MS. We generated 590 spectra from 84 different strains (including rare or atypical isolates) to cover this bacterial genus. We then applied a novel biomathematical approach to discriminate different species. This allowed accurate identification of Brucella isolates at the genus level with no misidentifications, in particular as the closely related and less pathogenic Ochrobactrum genus. The main zoonotic species (B. melitensis, B. abortus and B. suis) could also be identified at the species level with an accuracy of 100%, 92.9% and 100%, respectively. This MALDI-TOF reference database will be the first Brucella database validated for diagnostic and accessible to all VITEK MS users in routine. This will improve the diagnosis and control of brucellosis by allowing a rapid identification of these pathogens.

Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

Rates of processing determine the immunogenicity of immunoproteasome-generated epitopes.

CD8 T cells resolve intracellular pathogens by responding to pathogen-derived peptides that are presented on the cell surface by MHC class I molecules. Although most pathogens encode a large variety of antigenic peptides, protective CD8 T cell responses target usually only a few of these. To determine the mechanism by which the IFN-gamma-inducible proteasome (immuno) subunits enhance the ability of specific pathogen-derived peptides to elicit CD8 T cell responses, we generated a recombinant Listeria monocytogenes strain (rLM-E1) that secretes a model Ag encompassing the immunoproteasome-dependent E1B(192-200) and immunoproteasome-independent E1A(234-243) epitope. Analyses of Ag presentation showed that infected gene-deficient professional APCs, lacking the immunosubunits LMP7/ibeta5 and MECL-1/ibeta2, processed and presented the rLM-E1-derived E1B(192-200) epitope but with delayed kinetics. E1A epitope processing proceeded normally in these cells. Accordingly, infected gene-deficient mice failed to respond to the otherwise immunodominant E1B(192-200) epitope but mounted normal CD8 T cell responses to E1A(234-243) which was processed by the same professional APCs, from the same rLM-E1 Ag. The inability of gene-deficient mice to respond to E1B(192-200) was not explained by insufficient quantities of antigenic peptide, as splenic APC of 36-h-infected gene-deficient mice that presented the two E1 epitopes at steady state levels elicited responses to both E1B(192-200) and E1A(234-243) when transferred into LMP7+MECL-1-deficient mice. Taken together, our findings indicate that not absolute epitope quantities but early Ag-processing kinetics determine the ability of pathogen-derived peptides to elicit CD8 T cell responses, which is of importance for rational T cell vaccine design.

Role of Mycobacterium tuberculosis Ser/Thr kinase PknF: implications in glucose transport and cell division.

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB.

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.