Miguel Ángel Bratos Pérez

Procedure for microbial identification based on Matrix‐Assisted Laser Desorption/Ionization‐Time of Flight Mass Spectrometry from screening‐positive urine samples

Matrix‐Assisted Laser Desorption/Ionization‐Time of Flight Mass Spectrometry is a widely used proteomic technique in clinical microbiology laboratories, and enables microbial identification directly from clinical samples. This study seeks to establish a protocol for bacterial identification from monomicrobial urine samples that have tested positive in the screening with Sysmex UF‐1000i (Sysmex Corporation, Kobe, Japan). Sysmex UF‐1000i counts ≥1 × 107 bacteria/mL indicate a sufficient bacterial concentration to allow direct identification from urine, with 87.5% sensitivity. Microbial identification from urine with Sysmex UF‐1000i counts between 1 × 105 and 1 × 107 bacteria/ml requires preincubation to obtain the adequate amount of bacteria needed for analysis, and 91.7% sensitivity thus being achieved.

New procedure for rapid identification of microorganisms causing urinary tract infection from urine samples by mass spectrometry (MALDI-TOF)

INTRODUCTION Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry is widely established as a technique in clinical microbiology laboratories for the identification of microorganisms. Using this technique, it is also possible to obtain the identification of microorganisms from untreated urine samples. METHODS In this study, a differential centrifugation protocol and a criterion for validation of the results in order to achieve microbial identification from untreated urine samples are proposed. Additionally, the sensitivity of the analytical procedure in monobacterial urine samples has been evaluated. RESULTS A 90% sensitivity (confidence interval of 81.96%-94.84%) was obtained in urine samples with bacterial counts of ≥1×10(5)CFU/ml, and it was possible to improve the percentages of direct identifications from urine samples with bacterial counts of <1×10(5)CFU/ml. CONCLUSION It is concluded that the MALDI-TOF system is both fast and reliable in the identification of individual microorganisms from untreated urine samples with counts of ≥1×10(5)CFU/ml.

A two-hour antibiotic susceptibility test by ATP-bioluminescence

The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours.

Antibiograma rápido en Microbiología Clínica

The most widely used antibiotic susceptibility testing methods in Clinical Microbiology are based on the phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. These conventional methods take typically 24hours to obtain results. A review is presented here of recently developed techniques for the rapid determination of antibiotic susceptibility. Data obtained with different methods such as molecular techniques, flow cytometry, chemiluminescence, mass spectrometry, commercial methods used in routine work, colorimetric methods, nephelometry, microarrays, microfluids, and methods based on cell disruption and sequencing, are analyzed and discussed in detail.