Performance of MALDI-TOF MS, real-time PCR, antigen detection, and automated biochemical testing for the identification of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease highly endemic to Southeast Asia and northern Australia, though the area of endemicity is expanding. Cases may occur in returning travelers or, rarely, from imported contaminated products. Identification of B. pseudomallei is challenging for laboratories that do not see this organism frequently, and misidentifications by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and automated biochemical testing have been reported. The in vitro diagnostic database for use with the Vitek MS has recently been updated to include B. pseudomallei and we aimed to validate the performance for identification in comparison to automated biochemical testing with the Vitek 2 GN card, quantitative real-time polymerase chain reaction (qPCR) targeting the type III secretion system, and capsular polysaccharide antigen detection using a lateral flow immunoassay (LFA). We tested a "derivation" cohort including geographically diverse B. pseudomallei and a range of closely related Burkholderia species, and a prospective "validation" cohort of B. pseudomallei and B. cepacia complex clinical isolates. MALDI-TOF MS had a sensitivity of 1.0 and specificity of 1.0 for the identification and differentiation of B. pseudomallei from related Burkholderia species when a certainty cutoff of 99.9% was used. In contrast, automated biochemical testing for B. pseudomallei identification had a sensitivity of 0.83 and specificity of 0.88. Both qPCR and LFA correctly identified all B. pseudomallei isolates with no false positives. Due to the high level of accuracy, we have now incorporated MALDI-TOF MS into our laboratory's B. pseudomallei identification workflow.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a disease associated with high morbidity and mortality that disproportionately affects rural areas in Southeast Asia and northern Australia. The known area of endemicity is expanding and now includes the continental United States. Laboratory identification can be challenging which may result in missed or delayed diagnoses and poor patient outcomes. In this study, we compared mass spectrometry using an updated spectral database with multiple other methods for B. pseudomallei identification and found mass spectrometry highly accurate. We have therefore incorporated this fast and cost-effective method into our laboratory's workflow for B. pseudomallei identification.

Identification and antifungal susceptibility profile of uncommon yeast species at Fattouma Bourguiba University Hospital in Tunisia.

Despite the severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba University Hospital using phenotypic, molecular, and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua, and Candida guilliermondii. A total of 90% of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST, respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool.

There is still little information on the epidemiology of uncommon emergent yeasts, although their implication in severe diseases and mainly invasive infections. Thus, the importance of an accurate identification and antifungal susceptibility testing for a better monitoring of related infections.

Rapid and Nondestructive Evaluation of Wheat Chlorophyll under Drought Stress Using Hyperspectral Imaging.

Chlorophyll drives plant photosynthesis. Under stress conditions, leaf chlorophyll content changes dramatically, which could provide insight into plant photosynthesis and drought resistance. Compared to traditional methods of evaluating chlorophyll content, hyperspectral imaging is more efficient and accurate and benefits from being a nondestructive technique. However, the relationships between chlorophyll content and hyperspectral characteristics of wheat leaves with wide genetic diversity and different treatments have rarely been reported. In this study, using 335 wheat varieties, we analyzed the hyperspectral characteristics of flag leaves and the relationships thereof with SPAD values at the grain-filling stage under control and drought stress. The hyperspectral information of wheat flag leaves significantly differed between control and drought stress conditions in the 550-700 nm region. Hyperspectral reflectance at 549 nm (r = -0.64) and the first derivative at 735 nm (r = 0.68) exhibited the strongest correlations with SPAD values. Hyperspectral reflectance at 536, 596, and 674 nm, and the first derivatives bands at 756 and 778 nm, were useful for estimating SPAD values. The combination of spectrum and image characteristics (L*, a*, and b*) can improve the estimation accuracy of SPAD values (optimal performance of RFR, relative error, 7.35%; root mean square error, 4.439; R2, 0.61). The models established in this study are efficient for evaluating chlorophyll content and provide insight into photosynthesis and drought resistance. This study can provide a reference for high-throughput phenotypic analysis and genetic breeding of wheat and other crops.

Identification and analysis of pathogenic bacteria causing outbreaks in Indian major carp aquaculture of Tripura.

OBJECTIVE: The objective of this study was to investigate bacterial disease outbreaks in Indian major carp from aquaculture systems in Tripura, India, and identify the bacterial species associated with those outbreaks. METHODS: A 3-year surveillance was conducted in eight districts of Tripura, during which nine bacterial disease outbreaks were recorded. Fourteen bacterial strains isolated from diseased Indian major carp were selected and identified using phenotypic, molecular (16S ribosomal RNA gene), and phylogenetic analyses. In vitro pathogenicity studies were performed to assess the potential pathogenicity of the isolated bacteria. RESULT: The selected isolated strains were preliminarily identified under the genera Aeromonas (9 isolates), Acinetobacter (1 isolate), Citrobacter (3 isolates), and Pseudomonas (1 isolate). Molecular and phylogenetic analyses confirmed the species of the isolated bacteria, including Aeromonas jandaei (strains COF_AHE09 and COF_AHE61), Aeromonas veronii (strains COF_AHE13, COF_AHE52, COF_AHE55, COF_AHE56, and COF_AHE62), Aeromonas hydrophila (strains COF_AHE51 and COF_AHE58), Acinetobacter pittii (strain COF_AHE14), Citrobacter freundii (strains COF_AHE20, COF_AHE57, and COF_AHE59), and Pseudomonas aeruginosa (strain COF_AHE54). Behavioral and clinical signs observed in the diseased fish, such as lethargy, skin hemorrhaging, ulcers, fin and tail rot, exophthalmia, distended abdomen, scale loss, and skin discoloration, indicated the presence of bacterial septicemia. The in vitro pathogenicity studies highlighted the potential role of these bacteria in disease development, especially under environmental stress. CONCLUSION: This study provides valuable insights into the diversity of bacterial species associated with bacterial disease outbreaks in Indian major carp from aquaculture systems in Tripura. It serves as the first comprehensive investigation of its kind, contributing to our understanding of bacterial infections in Indian major carp.

Validation of the Colibrí Instrument for Automated Preparation of MALDI-TOF MS Targets for Yeast Identification.

Recently, Copan (Italy) introduced the Colibrí instrument for automated colony picking and preparation of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) target plates. Our study aimed to validate this system for yeasts as such testing has not been performed yet and is a missing link needed to implement the system for routine use. Fifty-five Candida strains were selected to evaluate the accuracy of Colibrí. For each strain, a sheep blood agar plate supplemented with X and V factors (HEM) and a Sabouraud agar plate (SAB) were inoculated and incubated using the WASPlab specimen processing system (Copan). After 18 h and 36 h of incubation, the isolates were spotted in parallel using Colibrí and manually onto MALDI-TOF target plates with the addition of formic acid and identified using MALDI-TOF mass spectrometry. The reproducibility was evaluated using ATCC reference and clinical isolate-derived strains. The cumulative percentage of acceptable identification scores (IDs) after 36 h was 91% for strains cultured on HEM plates using both Colibrí and the manual method. The SAB plates showed inferior results for both Colibrí (76%) and the manual method (78%). We observed an overall agreement of 92% at 18 h for identification of the strains on the HEM plates between Colibrí and the manual method and 94% after 36 h. For the SAB plates, the agreement was 78% after 18 h and 84% after 36 h. Apart from Candida dubliniensis and Candida tropicalis, all Candida species were identified with 100% accuracy using Colibrí on HEM plates. We observed good agreement between Colibrí and the manual reference method. These results demonstrate that Colibrí is a reliable system for MALDI-TOF target preparation for yeast identification, allowing increased standardization and less hands-on time.

Candida auris and some Candida parapsilosis strains exhibit similar characteristics on CHROMagarTMCandida Plus.

Candida auris is considered a public health problem due to its resistance and its tendency to cause nosocomial outbreaks. CHROMagarTMCandida Plus has recently been marketed as capable of presumptively identifying C. auris. The objective of this work was to analyze the ability of this new chromogenic medium to differentiate C. auris from other members of the C. haemulonii complex and from other yeasts commonly isolated in clinical practice. A collection of 220 strains including species of the C. haemulonii (n = 83) and C. parapsilosis (n = 80) complexes was studied. The strains were identified by molecular methods and cultured as individual or as mixed aqueous inoculum on CHROMagarTMCandida Plus plates. Colony morphotypes were evaluated at 5 time points. CHROMagarTMCandida Plus was a helpful tool for presumptive identification for C. auris. Better reading results were obtained after 48 hours of incubation at 35°C. It is able to easily differentiate C. auris from other closely related species of the C. haemulonii complex and other yeasts. This chromogenic medium would be also useful as screening and surveillance tool for C. auris colonization. However, we demonstrated that it would be a possible misidentification of C. parapsilosis as C. auris (44.3% showed similar morphotypes). To reduce false positives when it is used in a context of a C. auris outbreak, we propose to supplement the chromogenic medium with 8 µg/ml fluconazole. This modified medium was tested and it clearly differentiate C. parapsilosis from C. auris.

CHROMagarTMCandida Plus is able to differentiate C. auris from other Candida spp., including other species of the C. haemulonii complex. However, 44.3% of the tested C. parapsilosis strains would be misidentified as C. auris. We propose the addition of 8 µg/ml fluconazole to solve this issue.

Systematic Review of Mutations Associated with Isoniazid Resistance Points to Continuing Evolution and Subsequent Evasion of Molecular Detection, and Potential for Emergence of Multidrug Resistance in Clinical Strains of Mycobacterium tuberculosis.

Molecular testing is rapidly becoming an integral component of global tuberculosis (TB) control. Uncommon mechanisms of resistance escape detection by these platforms and undermine our ability to contain outbreaks. This article is a systematic review of published articles that reported isoniazid (INH) resistance-conferring mutations between September 2013 and December 2019. The genes katG, inhA, and fabG1, and the intergenic region oxyR'-ahpC were considered in this review. Fifty-two articles were included that described 9,306 clinical isolates (5,804 INH resistant [INHr] and 3,502 INH susceptible [INHs]) from 31 countries. The three most frequently mutated loci continue to be locus 315 of katG (katG315; n = 4,271), locus -15 of inhA (inhA-15; n = 787), and locus -8 of inhA (inhA-8; 106). However, the diagnostic value of inhA-8 is far lower than previously thought, as it only appears in 25 (0.4%) of the INHr isolates lacking the first two mutations. I catalogued 45 new loci (29 katG, nine inhA, and seven ahpC) associated with INH resistance and identified 59 loci (common to this and previous reviews) as a reliable basis for molecular diagnostics. Including all observed mutations provides a cumulative sensitivity of 85.6%. In 14.4% of resistant isolates, no mechanism of resistance was detected, making them likely to escape molecular detection, and in the case of INH monoresistance, likely to convert to multidrug-resistant TB (MDR-TB). Integrating the information cataloged in this study into current diagnostic tools is essential for combating the emergence of MDR-TB, and its exclusion can lead to an unintended selection against common mechanisms and to diversifying evolution. Observation of many low-frequency resistance-conferring mutations points to an advantage of whole-genome sequencing (WGS) for diagnostics. Finally, I provide five recommendations for future diagnostic platforms.

Genetic characteristics and ploidy trigger the high inducibility of double haploid (DH) inducer in Brassica napus.

BACKGROUND: Our recently reported doubled haploid (DH) induction lines e.g., Y3380 and Y3560 are allo-octoploid (AAAACCCC, 2n = 8× ≈ 76), which can induce the maternal parent to produce DH individuals. Whether this induction process is related to the production of aneuploid gametes form male parent and genetic characteristics of the male parent has not been reported yet. RESULTS: Somatic chromosome counts of DH inducer parents, female wax-less parent (W1A) and their F1 hybrid individuals revealed the reliability of flow cytometry analysis. Y3560 has normal chromosome behavior in metaphase I and anaphase I, but chromosome division was not synchronized in the tetrad period. Individual phenotypic identification and flow cytometric fluorescence measurement of F1 individual and parents revealed that DH individuals can be distinguished on the basis of waxiness trait. The results of phenotypic identification and flow cytometry can identify the homozygotes or heterozygotes of F1 generation individuals. The data of SNP genotyping coupled with phenotypic waxiness trait revealed that the genetic distance between W1A and F1 homozygotes were smaller as compared to their heterozygotes. It was found that compared with allo-octoploids, aneuploidy from allo-octoploid segregation did not significantly increase the DH induction rate, but reduced male infiltration rate and heterozygous site rate of induced F1 generation. The ploidy, SNP genotyping and flow cytometry results cumulatively shows that DH induction is attributed to the key genes regulation from the parents of Y3560 and Y3380, which significantly increase the induction efficiency as compared to ploidy. CONCLUSION: Based on our findings, we hypothesize that genetic characteristics and aneuploidy play an important role in the induction of DH individuals in Brassca napus, and the induction process has been explored. It provides an important insight for us to locate and clone the genes that regulate the inducibility in the later stage.

Unveiling the Virulent Genotype and Unusual Biochemical Behavior of Escherichia coli ST59.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of human and animal infections worldwide. The utilization of selective and differential media to facilitate the isolation and identification of E. coli from complex samples, such as water, food, sediment, and gut tissue, is common in epidemiological studies. During a surveillance study, we identified an E. coli strain isolated from human blood culture that displayed atypical light cream-colored colonies in chromogenic agar and was unable to produce ß-glucuronidase and ß-galactosidase in biochemical tests. Genomic analysis showed that the strain belongs to sequence type 59 (ST59) and phylogroup F. The evaluation in silico of 104 available sequenced lineages of ST59 complex showed that most of them belong to serotype O1:K1:H7, are ß-glucuronidase negative, and harbor a virulent genotype associated with the presence of important virulence markers such as pap, kpsE, chuA, fyuA, and yfcV. Most of them were isolated from extraintestinal human infections in diverse countries worldwide and could be clustered/subgrouped based on papAF allele analysis. Considering that all analyzed strains harbor a virulent genotype and most do not exhibit biochemical behavior typical of E. coli, we report that they could be misclassified or underestimated, especially in epidemiological studies where the screening criteria rely only on typical biochemical phenotypes, as happens when chromogenic media are used. IMPORTANCE The use of selective and differential media guides presumptive bacterial identification based on specific metabolic traits that are specific to each bacterial species. When a bacterial specimen displays an unusual phenotype in these media, this characteristic may lead to bacterial misidentification or a significant delay in its identification, putting a patient at risk depending on the infection type. In the present work, we describe a virulent E. coli sequence type (ST59) that does not produce beta-glucuronidase (GUS negative), production of which is the metabolic trait widely used for E. coli presumptive identification in diverse differential media. The recognition of this unusual metabolic trait may help in the proper identification of ST59 isolates, the identification of their reservoir, and the evaluation of the frequency of these pathogens in places where automatic identification methods are not available.

Comparing the phenotypic, genotypic, and proteomic identification of Trichosporon species: A globally emerging yeast of medical importance.

Trichosporon spp. are widely distributed in the nature, comprising species that inhabit different ecological niches and can be found in the water, soil, and body surface of animals and humans. Such microorganisms have been classically associated with superficial infections; however, in the last decades, they have also been related to disseminated infections in immunocompromised patients, behaving as opportunistic agents, which demands rapid and accurate species identification for efficient therapy. Concordance level between the traditional phenotypic method and the molecular technique (gold standard) in the identification of all 59 Trichosporon samples was 59.3%. Identification concordance between MALDI-TOF spectrometry and the molecular technique was 71.2%. No isolate of environmental origin was identifiable by MALDI-TOF mass spectrometry (MS), and 100% of such environmental isolates were discordant for IGS region sequencing and phenotypic characterization. Both comparisons evidenced greatest concordance in the identification of T. asahii. The species T. debeurmannianum, T. dermatis, T. venhuisii and T. insectorum were not properly identified by both MALDI-TOF MS and the phenotypic technique. MALDI-TOF MS, in particular, seems to be appropriate to investigate yeasts of the genus Trichosporon; however, database updates are still necessary, especially for species that are not common in the clinical routine. With the aim of helping understand the aspects involved in early and accurate diagnosis of infections caused by this opportunistic agent, the present study compared the phenotypic, molecular (IGS region) and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon which had clinical and environmental origin and were kept in a mycology collection.

The present study compared the phenotypic, genotypic, and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon. MALDI-TOF MS, in particular, seems to be appropriate to investigate this yeasts when compared to a molecular technique (gold standard).

Clinical and Biological Features of Cutibacterium (Formerly Propionibacterium) avidum, an Underrecognized Microorganism.

The recent description of the genus Cutibacterium has altered the taxonomy of Propionibacterium species. These organisms still belong to the genera of the skin coryneform group, and the most-studied species remains Cutibacterium acnes. Cutibacterium avidum is also a known skin commensal. This underrecognized microorganism can, however, act as a pathogen after bacterial seeding and can be considered opportunistic, causing either superficial or deep/invasive infections. It can cause numerous infections, including but not limited to breast infections, skin abscesses, infective endocarditis, and device-related infections. The ecological niche of C. avidum is clearly different from that of other members of the genus: it is found in the axillary region or at wet sites rather than in dry, exposed areas, and the number of microorganisms increases during puberty. Historically, it has been used for its ability to modulate the immune response and for its antitumor properties. Conventional microbial culture methods and identification processes allow for its accurate identification and characterization. Thanks to the modern omics tools used for phylogenomic approaches, understanding C. avidum pathogenesis (including host-bacterium interactions and virulence factor characterization) is becoming easier, allowing for more thorough molecular characterization. These analyses have revealed that C. avidum causes diverse diseases mediated by multiple virulence factors. The recent genome approach has revealed specific genomic regions within this species that are involved in adherence and biofilm formation as well as fitness, survival, and defense functions. Numerous regions show the presence of phages and horizontal gene transfer. C. avidum remains highly sensitive to a broad spectrum of antibiotics, such as ß-lactams, fluoroquinolones, macrolides, and rifampin, although erythromycin and clindamycin resistance has been described. A long-term treatment regimen with a combination of antibiotics is required to successfully eliminate the remaining adherent bacteria, particularly in the case of deep infections after debridement surgery.

Identification of Candida auris by Use of the Updated Vitek 2 Yeast Identification System, Version 8.01: a Multilaboratory Evaluation Study.

Candida auris is an emerging multidrug-resistant yeast that has been systematically incorrectly identified by phenotypic methods in clinical microbiology laboratories. The Vitek 2 automated identification system (bioMérieux) recently included C. auris in its database (version 8.01). We evaluated the performance of the Vitek 2 YST ID card to identify C. auris and related species. A panel of 44 isolates of Candida species (C. auris, n = 35; Candida haemulonii, n = 5; Candida duobushaemulonii, n = 4) were tested by three different hospital-based microbiology laboratories. Among 35 isolates of C. auris, Vitek 2 yielded correct identification in an average of 52% of tested samples. Low-discrimination (LD) results with an inability to distinguish between C. auris, C. duobushaemulonii, and Candida famata were obtained in an average of 27% of samples. Incorrect identification results were obtained in an average of 21% of samples, the majority (91%) of which were reported as C. duobushaemulonii and the remaining 9% of which were reported as Candida lusitaniae/C. duobushaemulonii. The proportion of correct identification was not statistically different across different centers (P = 0.78). Stratification by genetic clades demonstrated that 100% (n = 8) of the strains of the South American clade were correctly identified compared to 7% (n = 10) and 0% (n = 4) from the African and East Asian clades, respectively. None of the non-auris Candida strains (n = 9) were incorrectly identified as C. auris Our results show that the Vitek 2 (version 8.01) yeast identification system has a limited ability to correctly identify C. auris These data suggest that an identification result for C. duobushaemulonii should warrant further testing to rule out C. auris The overall performance of the Vitek 2 seems to differ according to C. auris genetic clade, with the South American isolates yielding the most accurate results.

Typeability of MALDI-TOF assay for identification of non-aureus staphylococci associated with bovine intramammary infections and teat apex colonization.

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), a culture-dependent assay, has recently been implemented for routine identification of non-aureus staphylococci (NAS) species from milk, but the assay has never been investigated for NAS from nonmilk or environmental samples. The objective of this study was to evaluate the typeability of the MALDI-TOF assay for the identification and differentiation of bovine-associated NAS species on aseptically collected quarter milk and teat skin samples in dairy herds. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabs and foremilk samples from right hind and left front quarters. Teat skin swabs and milk samples were collected aseptically for preliminary identification using bacterial culture on chromogenic and calf blood agars. Colonies from milk and teat skin samples with suspicion of having NAS were identified to species-level by MALDI-TOF assay. Out of 511 isolates from 284 quarters (142 cows), 78% (n = 399) were identified by MALDI-TOF. The percentage of correctly identified NAS from milk (91%, 105/115) using MALDI-TOF was higher than the percentage from teat skin (68%, 268/396). Out of the identified isolates, 93% (n = 373) were successfully identified as NAS, whereas the remaining 26 (7%) were shown to be other bacterial species. Out of 26 NAS isolates, 1 originated from milk (Corynebacterium stationis), whereas 25 originated from teat skin representing Aerococcus viridans (n = 7), Bacillus pumilus (n = 13), Enterococcus saccharolyticus (n = 1), Clostridium septicum (n = 1), Corynebacterium stationis (n = 2), and Corynebacterium casei (n = 1). The MALDI-TOF identified 85 (98/115) and 62% (245/396) of the isolates in the first test. Isolates that were not identified to species-level at first test were subjected to a second test, and 47 (8/17) and 32% (48/151) from milk and teat skin, respectively, were identified. After 2 rounds of MALDI-TOF, 22% (n = 112) of the isolates were not identified, representing 103 from teat skin and 9 from milk. Eighteen isolates without identification by MALDI-TOF were successfully identified to species-level using sequencing, where 16 were correctly identified as NAS, whereas the other 2 were Corynebacterium stationis. In conclusion, MALDI-TOF is a reliable assay for identification and typeability of NAS species from aseptically collected quarter milk samples. The assay may be used for identification of NAS species from teat skin swabs. However, confirmation using nucleic acid-based tools is vital for accurate species identification of some species and strains.

Multilocus Phylogeny and Antifungal Susceptibility of Aspergillus Section Circumdati from Clinical Samples and Description of A. pseudosclerotiorum sp. nov.

A multilocus phylogenetic study was carried out to assess species identity of a set of 34 clinical isolates from Aspergillus section Circumdati from the United States and to determine their in vitro antifungal susceptibility against eight antifungal drugs. The genetic markers used were the internal transcribed spacer (ITS) region, and fragments of the beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes. The drugs tested were amphotericin B, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, and terbinafine. The most common species sampled was A. westerdijkiae (29.4%), followed by a novel species, which was described here as A. pseudosclerotiorum (23.5%). Other species identified were A. sclerotiorum (17.6%), A. ochraceus (8.8%), A. subramanianii (8.8%), and A. insulicola and A. ochraceopetaliformis, with two isolates (5.9%) of each. The drugs that showed the most potent activity were caspofungin, micafungin, and terbinafine, while amphotericin B showed the least activity.

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.

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media.

During the last decade, many investigators have studied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of mycobacteria. Diverse and contradictory results indicated that optimal level for routine testing has not been reached yet. This work aimed to assess Vitek MS through two distinct versions, Saramis v4.12 RUO and the IVD v3.0, under conditions close to routine laboratory practice. Overall, 111 mycobacterial isolates were subjected to protein extraction and same spectra were matched against both databases. The IVD v3.0 database proved to be superior to Saramis v4.12 and its identification rates remarkably increased, from 67% to 94% for isolates grown on Middlebrook 7H10 solid medium and from 62% to 91% for isolates grown on mycobacterial growth indicator tube (MGIT) liquid medium. With this new version, IVD v3.0, MALDI-TOF MS might be integrated into routine clinical diagnostics, although molecular techniques remain mandatory in some cases.

First record of the rare species Aeromonas schubertii from mussels: phenotypic and genetic reevaluation of the species and a review of the literature.

In a study where the prevalence of Aeromonas in shellfish was analysed, three isolates of Aeromonas schubertii were identified, representing this the first report of this species from mussels. This species was originally described in 1988 from strains isolated from extra-intestinal human infections and since then has been cited in only 18 occasions. For many years, A. schubertii was the only mannitol-negative species of the genus. However, three additional mannitol-negative species (Aeromonas simiae, Aeromonas diversa and Aeromonas australiensis) have been described. This, together with the fact that A. schubertii is a rare human pathogenic species, motivated the present study to characterize its biochemical behaviour and differentiation from the other mannitol-negative species. The molecular similarity (16S rRNA, rpoD and gyrB genes) of the strains, presence of virulence genes and antimicrobial resistance were determined. All A. schubertii strains showed the same phenotypic behaviour, i.e. they use citrate, are positive for lysine decarboxylase and DL-lactate, but negative for production of mannitol, indole and acid from sucrose and could be easily differentiated from other mannitol-negative species. All strains carried the aerA and lafA virulence genes and showed susceptibility to all antibiotics tested. Seafood could be a transmission route of this bacterium to humans.

Comparison of methods for the identification of microorganisms isolated from blood cultures.

BACKGROUND: Bloodstream infections are responsible for thousands of deaths each year. The rapid identification of the microorganisms causing these infections permits correct therapeutic management that will improve the prognosis of the patient. In an attempt to reduce the time spent on this step, microorganism identification devices have been developed, including the VITEK(®) 2 system, which is currently used in routine clinical microbiology laboratories. METHODS: This study evaluated the accuracy of the VITEK(®) 2 system in the identification of 400 microorganisms isolated from blood cultures and compared the results to those obtained with conventional phenotypic and genotypic methods. In parallel to the phenotypic identification methods, the DNA of these microorganisms was extracted directly from the blood culture bottles for genotypic identification by the polymerase chain reaction (PCR) and DNA sequencing. RESULTS: The automated VITEK(®) 2 system correctly identified 94.7 % (379/400) of the isolates. The YST and GN cards resulted in 100 % correct identifications of yeasts (15/15) and Gram-negative bacilli (165/165), respectively. The GP card correctly identified 92.6 % (199/215) of Gram-positive cocci, while the ANC card was unable to correctly identify any Gram-positive bacilli (0/5). CONCLUSIONS: The performance of the VITEK(®) 2 system was considered acceptable and statistical analysis showed that the system is a suitable option for routine clinical microbiology laboratories to identify different microorganisms.

Fungal keratitis due to Schizophyllum commune: an emerging pathogenic fungus.

Fungal keratitis due to Schizophyllum commune is very rare. In this study, we report the clinical and microbiological profile of five patients with fungal keratitis due to S. commune. Direct microscopic examination of corneal scrapings from all five patients showed septate branching hyaline fungal filaments. Similarly, in all five patients Sabouraud dextrose agar (SDA) plates inoculated with corneal scrapings showed white, cottony colonies on the second day of incubation. Lactophenol cotton blue stained wet preparation of 7-day-old colonies on SDA revealed clamp connections and no spores. The fungus was identified by its characteristic clamp connections, fan-shaped bracket fruiting body with pinkish-grey longitudinally split-radiating gills. The phenotypic identification of one of the five isolates further conformed by ITS sequencing. Treatment outcome was available for two of the five patients; in these two patients, the keratitis resolved with topical natamycin.