Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) recently moved from physics laboratories into diagnostic clinical microbiology.In this capacity it is currently replacing phenotypic bacterial identification tools that were designed and developed by Pasteur and Koch.Many European microbiology laboratories now use MALDI-TOF-MS with a high degree of efficiency.In the USA this will soon become common as well since the Food and Drug Administration (FDA) has now cleared the first of the commercially available systems for in vitro diagnostic use.This will also further spark the development of mass spectrometry applications in the field of antimicrobial susceptibility testing and epidemiological typing.This review summarizes the current state of affairs with respect to MALDI-TOF-MS in clinical microbiology.Emphasis will be on clinical relevance and studies in that field and the diagnostic data obtained through comparative analyses of different MALDI-TOF-MS instrumentation and through multi-center validation studies will be reviewed.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement.
Classification ; Mass Spectrometry* ; Methods

Objeetive:To update the status of Gardnerella vaginalis (G.vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology,metronidazole resistance and virulence properties.Methods:It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur,Malaysia) prospective study with laboratory-based microbiological follow up and analyses.Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis,isolation and identification of G.vaginalis,metronidazole susceptibility testing,vaginolysin and sialidase gene PCR,Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping.Results:Among the 207 patients suspected for BV,G.vaginalis was isolated from 47 subjects.G.vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples.Three G.vaginalis isolates were resistant to metronidazole.Biotyping revealed 1 and 7 as the common types.Amplified ribosomal DNA restriction analysis genotype Ⅱ was found to be more common (n =22;46％) than Ⅰ (n =12;25.53％) and Ⅲ (n =13;27.6％).All genotype Ⅰ and Ⅲ isolates carried the sialidase gene,while 91.6％ and 84.6％ contained the vaginolysin gene.Genotype Ⅰ was significantly associated with postgynaecological surgical complications and abortions (P =0.002).Conclusions:The existence of pathogenic G.vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.

Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted "revolution" was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches.
Bacteria/*genetics/isolation & purification/metabolism ; Bacterial Infections/diagnosis/*microbiology ; Electronic Nose ; High-Throughput Nucleotide Sequencing ; Humans ; Mass Spectrometry ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques

The prevalence of multidrug-resistant gram-negative bacteria has continuously increased over the past few years; bacterial strains producing AmpC β-lactamases and/or extended-spectrum β-lactamases (ESBLs) are of particular concern. We combined high-resolution whole genome sequencing and phenotypic data to elucidate the mechanisms of resistance to cephamycin and β-lactamase in Korean Klebsiella pneumoniae strains, in which no AmpC-encoding genes were detected by PCR. We identified several genes that alone or in combination can potentially explain the resistance phenotype. We showed that different mechanisms could explain the resistance phenotype, emphasizing the limitations of the PCR and the importance of distinguishing closely-related gene variants.
Drug Resistance, Microbial ; Genome ; Gram-Negative Bacteria ; Klebsiella pneumoniae* ; Klebsiella* ; Korea* ; Phenotype ; Polymerase Chain Reaction ; Prevalence

Objective To update the status of Gardnerella vaginalis (G. vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology, metronidazole resistance and virulence properties. Methods It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur, Malaysia) prospective study with laboratory-based microbiological follow up and analyses. Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis, isolation and identification of G. vaginalis, metronidazole susceptibility testing, vaginolysin and sialidase gene PCR, Piot's biotyping and amplified ribosomal DNA restriction analysis (ARDRA) genotyping. Results Among the 207 patients suspected for BV, G. vaginalis was isolated from 47 subjects. G. vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples. Three G. vaginalis isolates were resistant to metronidazole. Biotyping revealed 1 and 7 as the common types. ARDRA genotype II was found to be more common (n = 22; 46%) than I (n = 12; 25.53%) and III (n = 13; 27.6%). All genotype I and III isolates carried the sialidase gene, while 91.6% and 84.6% contained the vaginolysin gene. Genotype I was significantly associated with post-gynaecological surgical complications and abortions (P = 0.002). Conclusions The existence of pathogenic G. vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.