Improved Differentiation of Streptococcus pneumoniae and Other S. mitis Group Streptococci by MALDI Biotyper Using an Improved MALDI Biotyper Database Content and a Novel Result Interpretation Algorithm.

Reliable distinction of Streptococcus pneumoniae and viridans group streptococci is important because of the different pathogenic properties of these organisms. Differentiation between S. pneumoniae and closely related Sreptococcusmitis species group streptococci has always been challenging, even when using such modern methods as 16S rRNA gene sequencing or matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. In this study, a novel algorithm combined with an enhanced database was evaluated for differentiation between S. pneumoniae and S. mitis species group streptococci. One hundred one clinical S. mitis species group streptococcal strains and 188 clinical S. pneumoniae strains were identified by both the standard MALDI Biotyper database alone and that combined with a novel algorithm. The database update from 4,613 strains to 5,627 strains drastically improved the differentiation of S. pneumoniae and S. mitis species group streptococci: when the new database version containing 5,627 strains was used, only one of the 101 S. mitis species group isolates was misidentified as S. pneumoniae, whereas 66 of them were misidentified as S. pneumoniae when the earlier 4,613-strain MALDI Biotyper database version was used. The updated MALDI Biotyper database combined with the novel algorithm showed even better performance, producing no misidentifications of the S. mitis species group strains as S. pneumoniae All S. pneumoniae strains were correctly identified as S. pneumoniae with both the standard MALDI Biotyper database and the standard MALDI Biotyper database combined with the novel algorithm. This new algorithm thus enables reliable differentiation between pneumococci and other S. mitis species group streptococci with the MALDI Biotyper.

Performance of MALDI-TOF MS for identification of oral Prevotella species.

During the past decade, the clinically relevant genus Prevotella has expanded considerably. Prevotella species can be isolated from nearly all types of oral infections but also from various non-oral infections. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in clinical microbiology laboratories as a convenient method for identifying bacterial isolates from clinical specimens. Here we tested the diagnostic accuracy of a total of 123 oral Prevotella isolates, selected based on their biochemical profile, by Bruker MALDI-TOF MS. Partial 16S rRNA sequencing was used as a reference method. The performance of MALDI-TOF MS to identify the isolates to the genus level was excellent with 100.0% accuracy, while a good identification rate of 88.6% was achieved to the species level with a log score of ≥2.0. The isolates representing P. aurantiaca and P. jejuni, which are currently missing from the MALDI BioTyper database, were identified correctly to the genus level. Of the 123 isolates, one P. pallens isolate (0.8%) was identified with a score variation of 1.7-1.999. Overall, biochemical testing produced a high proportion (70.7%) of incorrect identifications within different species. MALDI-TOF MS offers a reliable and rapid method for the identification of Prevotella species included in the database.

Factors affecting the quality and reproducibility of MALDI-TOF MS identification for human Capnocytophaga species.

Reproducibility and quality of MALDI-TOF MS spectra are critical in the identification process, however, information on the factors affecting the identification scores are scarce. Here, we studied the influence of various factors during the identification process of human oral Capnocytophaga species. The influence of two incubation times, plate-spotting reproducibility of two examiners, extraction technique, storage period of plates, and different laser repetition rates on the quality of MALDI-TOF MS identification of 34 human Capnocytophaga strains (including C. gingivalis, C. granulosa, C. haemolytica, C. leadbetteri, C. ochracea, C. sputigena, and Capnocytophaga genospecies AHN8471) was examined. The identification rate did not show a significant difference (P = 0.05) between the two incubation times, except that C. haemolytica needed a longer incubation time to be recognized at the genus level. The reproducibility of spotting between two examiners was ensured by following the manufacturer's instructions. At the species level, formic acid extraction improved the identification of species with limited representation in the database, such as C. haemolytica and C. granulosa. The storage of plates for one week decreased the identification scores. No significant difference (P = 0.39) was observed between the 60 Hz and 120 Hz laser repetition rates for identifying Capnocytophaga species to the genus or species level. In conclusion, the MALDI TOF MS offers a reliable Capnocytophaga identification after following the universal protocol, while the formic acid extraction is restricted to species with a limited number of strains in the database.

Brachybacterium sp. CH-KOV3 isolated from an oil-polluted environment-a new producer of levan.

Various microorganisms isolated from polluted environments, such as Pseudomonas sp. and Micrococcus sp. can synthesize exopolysaccharides (EPSs) which are natural, non-toxic and biodegradable polymers. EPSs play a key role in protection of microbial cells under various external influences. For humans, these substances have potential use in many industries. EPSs can be applied as a flavor or a fragrance carrier, an emulsifier, a stabilizer, a prebiotic, an antioxidant or an antitumor agent. In this study, we characterized an environmental microorganism that produces EPS, optimized EPS production by this strain and characterized the EPS produced. Isolate CH-KOV3 was identified as Brachybacterium paraconglomeratum. The sucrose level in the growth medium greatly influenced EPS production, and the highest yield was when the microorganism was incubated in media with 500g/L of sucrose. The optimal temperature and pH were 28°C and 7.0, respectively. The nuclear magnetic resonance (NMR) results and GC-MS analysis confirmed that the residues were d-fructofuranosyl residues with ß-configuration, where fructose units are linked by ß-2,6-glycosidic bonds, with ß-2,1-linked branches. All these data indicate that the investigated EPS is a levan-type polysaccharide. Thus, it was concluded that Brachybacterium sp. CH-KOV3 could constitute a new source for production of the bioactive polysaccharide, levan.

Scedosporium apiospermum as a rare cause of central skull base osteomyelitis.

We report a case of Scedosporium apiospermum mold causing ear infection, central skull base osteomyelitis and finally, occlusion of carotid artery in a 48-year-old diabetic man. The exact diagnosis was established and the severity of the disease understood several months after the onset of symptoms. Despite of appropriate antifungal therapy, and repeated surgical and otological procedures, the infection progressed to fatal cerebral infarction.

Use of an automated PCR assay, the GenomEra S. pneumoniae, for rapid detection of Streptococcus pneumoniae in blood cultures.

BACKGROUND: Streptococcus pneumoniae is recognized as a major cause of pneumonia, meningitis, and bacteremia. Since the mortality rate for pneumococcal bacteremia remains high, the reliable detection of the bacterium in blood samples is important. In this study, the performance of a new automated PCR assay, the GenomEra(™) S. pneumoniae, for direct detection of S. pneumoniae in blood cultures was investigated. METHODS: In total, 200 samples were analyzed, including 90 previously identified culture collection isolates and 110 blood culture specimens. The species identification was confirmed with routine diagnostic methods including MALDI-TOF or 16S rDNA sequencing. RESULTS: From culture collection, the GenomEra S. pneumoniae assay correctly identified all 37 S. pneumoniae isolates consisting of 18 different serotypes, while all 53 non-S. pneumoniae isolates yielded negative test results. Of 110 blood culture specimens, 46 grew S. pneumoniae and all were positive by the GenomEra assay direct from bottle. The detection sensitivity and specificity of the GenomEra assay for direct analysis of S. pneumoniae in signal positive blood culture bottles was 100%, respectively. With a straightforward sample preparation protocol of blood cultures the results were available within 55 min, thus being significantly quicker than by the routinely used identification methods (18-48 h). The two-step, time-resolved fluorometric measurement mode employed by the GenomEra CDX(™) instrument showed no interference from blood or charcoal. CONCLUSION: The GenomEra S. pneumoniae assay is a tool that performs well for the rapid and reliable detection of S. pneumoniae in blood cultures.