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.

Murine Model for Measuring Effects of Humanized-Dosing of Antibiotics on the Gut Microbiome.

There is a current need for enhancing our insight in the effects of antimicrobial treatment on the composition of human microbiota. Also, the spontaneous restoration of the microbiota after antimicrobial treatment requires better understanding. This is best addressed in well-defined animal models. We here present a model in which immune-competent or neutropenic mice were administered piperacillin-tazobactam (TZP) according to human treatment schedules. Before, during and after the TZP treatment, fecal specimens were longitudinally collected at established intervals over several weeks. Gut microbial taxonomic distribution and abundance were assessed through culture and molecular means during all periods. Non-targeted metabolomics analyses of stool samples using Quadrupole Time of Flight mass spectrometry (QTOF MS) were also applied to determine if a metabolic fingerprint correlated with antibiotic use, immune status, and microbial abundance. TZP treatment led to a 5-10-fold decrease in bacterial fecal viability counts which were not fully restored during post-antibiotic follow up. Two distinct, relatively uniform and reproducible restoration scenarios of microbiota changes were seen in post TZP-treatment mice. Post-antibiotic flora could consist of predominantly Firmicutes or, alternatively, a more diverse mix of taxa. In general, the pre-treatment microbial communities were not fully restored within the screening periods applied. A new species, closely related to Eubacterium siraeum, Mageeibacillus indolicus, and Saccharofermentans acetigenes, became predominant post-treatment in a significant proportion of mice, identified by 16S rRNA gene sequencing. Principal component analysis of QTOF MS of mouse feces successfully distinguished treated from non-treated mice as well as immunocompetent from neutropenic mice. We observe dynamic but distinct and reproducible responses in the mouse gut microbiota during and after TZP treatment and propose the current murine model as a useful tool for defining the more general post-antibiotic effects in the gastro-intestinal ecosystem where humanized antibiotic dosing may ultimately facilitate extrapolation to humans.

Developmental roadmap for antimicrobial susceptibility testing systems.

Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability, optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions.

Education of the PhD in laboratory medicine.

Throughout the history of laboratory medicine, the PhD scientist has played a role in developing new methods and algorithms that have contributed significantly to the field. Although the number of formally trained PhDs in laboratory medicine is currently small, they continue to play an important role in large, primarily academic, clinical laboratories and departments and in the in vitro diagnostics industry. This article discusses the importance of the formally trained PhD in today's laboratory medicine environment and the necessary training process, and approach for training PhDs at the postdoctoral level to have successful careers in laboratory medicine.

Eradication of methicillin-resistant Staphylococcus aureus from a neonatal intensive care unit by active surveillance and aggressive infection control measures.

OBJECTIVES: To describe an outbreak of hospital-acquired MRSA in a NICU and to identify the risk factors for, outcomes of, and interventions that eliminated it. SETTING: An 18-bed, level III-IV NICU in a community hospital. METHODS: Interventions to control MRSA included active surveillance, aggressive contact isolation, and cohorting and de-colonization of infants and HCWs with MRSA. A case-control study was performed to compare infants with and without MRSA. RESULTS: A cluster of 6 cases of MRSA infection between September and October 2001 represented an increased attack rate of 21.2% compared with 5.3% in the previous months. Active surveillance identified unsuspected MRSA colonization in 6 (21.4%) of 28 patients and 6 (5.5%) of 110 HCWs screened. They were all successfully decolonized. There was an increased risk of MRSA colonization and infection among infants with low birth weight or younger gestational age. Multiple gestation was associated with an increased risk of colonization (OR, 37.5; CI95, 3.9-363.1) and infection (OR, 5.36; CI95, 1.37-20.96). Gavage feeding (OR, 10.33; CI95, 1.28-83.37) and intubation (OR, 5.97; CI95, 1.22-29.31) were associated with increased risk of infection. Infants with MRSA infection had a significantly longer hospital stay than infants without MRSA (51.83 vs 21.46 days; P = .003). Rep-PCR with mec typing and PVL analysis confirmed the presence of a single common strain of hospital-acquired MRSA. CONCLUSION: Active surveillance, aggressive implementation of contact isolation, cohorting, and decolonization effectively eradicated MRSA from the NICU for 2 1/2 years following the outbreak.

In-house validation of the BACTEC 9240 blood culture system for detection of bacterial contamination in platelet concentrates.

BACKGROUND: At present, only two commercially available automated culture systems are cleared by the FDA for the purpose of quality control (QC) testing for bacterial contamination of platelet (PLT) concentrates: the BacT/ALERT blood culture system (bioMérieux) and the Pall eBDS (Pall Corporation), both of which allow testing of leukoreduced apheresis as well as whole blood-derived PLTs. After the decision of the AABB to institute universal QC testing of PLT concentrates for evidence of bacterial contamination, in-house validation of the performance of our current blood culture system, the BACTEC 9240, was carried out for this purpose. STUDY DESIGN AND METHODS: Serial dilutions of nine species of bacteria commonly associated with PLT contamination were prepared in one single-donor apheresis PLT unit per organism. Four mL of dilutions containing less than 1 to greater than 10(3) colony-forming units (CFUs) per mL was inoculated into blood culture bottles (Standard 10 Aerobic/F, Becton-Dickinson Diagnostic Systems) and incubated in a BACTEC 9240 continuously monitored blood culture system. Positive bottles were removed from the system and subcultured to insure the identity of bacterial growth. RESULTS: With the exception of Streptococcus mitis, the BACTEC system provided a detection sensitivity of less than 10 CFUs per mL for Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Enterobacter cloacae, and Pseudomonas aeruginosa. The limit of detection for the S. mitis test strain was 61 CFUs per mL. Detection of positive bottles ranged from 6.5 to 17.6 hours depending on the species tested and the cell density of the inoculum. Ongoing use of this system for bacterial detection yielded two true-positive samples from 3879 apheresis PLT products collected at our hospital-based donor center over 9 months. CONCLUSION: This study validates the use of the BACTEC 9240 continuously monitored blood culture system for the detection of low-level bacterial contamination in single donor apheresis PLTs in less than 24 hours.