Validation of Fourier Transform Infrared Spectroscopy for Serotyping of Streptococcus pneumoniae.

Fourier transform infrared (FT-IR) spectroscopy (IR Biotyper; Bruker) allows highly discriminatory fingerprinting of closely related bacterial strains. In this study, FT-IR spectroscopy-based capsular typing of Streptococcus pneumoniae was validated as a rapid, cost-effective, and medium-throughput alternative to the classical phenotypic techniques. A training set of 233 strains was defined, comprising 34 different serotypes and including all 24 vaccine types (VTs) and 10 non-vaccine types (NVTs). The acquired spectra were used to (i) create a dendrogram where strains clustered together according to their serotypes and (ii) train an artificial neural network (ANN) model to predict unknown pneumococcal serotypes. During validation using 153 additional strains, we reached 98.0% accuracy for determining serotypes represented in the training set. Next, the performance of the IR Biotyper was assessed using 124 strains representing 59 non-training set serotypes. In this setting, 42 of 59 serotypes (71.1%) could be accurately categorized as being non-training set serotypes. Furthermore, it was observed that comparability of spectra was affected by the source of the Columbia medium used to grow the pneumococci and that this complicated the robustness and standardization potential of FT-IR spectroscopy. A rigorous laboratory workflow in combination with specific ANN models that account for environmental noise parameters can be applied to overcome this issue in the near future. The IR Biotyper has the potential to be used as a fast, cost-effective, and accurate phenotypic serotyping tool for S. pneumoniae.

Aspergillus fumigatus and aspergillosis: From basics to clinics.

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

Phototransformations of 2-(1,2,4-Triazol-3-yl)benzoic Acid in Low Temperature Matrices.

Phototransformations of 2-(1,2,4-triazol-3-yl)benzoic acid induced by tunable UV laser were studied in low temperature matrices by FTIR spectroscopy. Out of 36 possible isomers of this molecule the most stable one, comprising the intramolecular N-H···O hydrogen bond, was detected experimentally in argon and xenon matrices after deposition. Upon irradiation with λ = 325 nm, two new conformers of the precursor were formed. The corresponding reverse photorotamerizations were also detected at 250 nm partly reproducing the initial molecules. In addition to the conformational changes, an interesting photoisomerization took place in the studied matrices leading to a proton transfer from N atom of the triazole ring to the carbonyl oxygen of the carboxylic group. As a result of this reaction, a new product with two OH groups was identified for the first time. The experimental studies were supported by DFT calculations in both ground and excited electronic states.

Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Validation of MALDI-TOF MS Biotyper database optimized for anaerobic bacteria: The ENRIA project.

Within the ENRIA project, several 'expertise laboratories' collaborated in order to optimize the identification of clinical anaerobic isolates by using a widely available platform, the Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Main Spectral Profiles (MSPs) of well characterized anaerobic strains were added to one of the latest updates of the Biotyper database db6903; (V6 database) for common use. MSPs of anaerobic strains nominated for addition to the Biotyper database are included in this validation. In this study, we validated the optimized database (db5989 [V5 database] + ENRIA MSPs) using 6309 anaerobic isolates. Using the V5 database 71.1% of the isolates could be identified with high confidence, 16.9% with low confidence and 12.0% could not be identified. Including the MSPs added to the V6 database and all MSPs created within the ENRIA project, the amount of strains identified with high confidence increased to 74.8% and 79.2%, respectively. Strains that could not be identified using MALDI-TOF MS decreased to 10.4% and 7.3%, respectively. The observed increase in high confidence identifications differed per genus. For Bilophila wadsworthia, Prevotella spp., gram-positive anaerobic cocci and other less commonly encountered species more strains were identified with higher confidence. A subset of the non-identified strains (42.1%) were identified using 16S rDNA gene sequencing. The obtained identities demonstrated that strains could not be identified either due to the generation of spectra of insufficient quality or due to the fact that no MSP of the encountered species was present in the database. Undoubtedly, the ENRIA project has successfully increased the number of anaerobic isolates that can be identified with high confidence. We therefore recommend further expansion of the database to include less frequently isolated species as this would also allow us to gain valuable insight into the clinical relevance of these less common anaerobic bacteria.

A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS.

Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories.

Metastases of renal clear cell carcinoma to ovary--case report and review of the literature.

In the literature the renal-ovarian axis has been demonstrated. Although, kidney and ovary are in a very distant anatomic position, they are supposed to have a lot of in common. This unusual connection begins from embryology, vascularization, and metastasizing tumors to each other. In the present systemic review the authors showed 24 case reports published in the literature, describing the metastases of primary renal cancer to ovary and only four cases reporting primary ovarian cancer metastases to kidney. Finding primary origin of the tumor is crucial in diagnostic process and subsequent therapy. The present case is a 25th case of renal cell carcinoma (RCC) metastasizing to ovary. The authors report the case of 51-year old woman with a four-year history of metastatic renal clear cell carcinoma (MRCC) presented in the present hospital with contralateral metastasis in right ovary.

Efficient identification of Malassezia yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. OBJECTIVES: To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS. METHODS: An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand. RESULTS: MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA. CONCLUSIONS: Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks.

Rapid detection of antibiotic resistance based on mass spectrometry and stable isotopes.

With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.

Identification of Haemophilus influenzae and Haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244 H. influenzae and 33 H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.

Carboxylic group and its tetrazolyl isostere in one molecule. Matrix isolation FTIR and DFT studies on thermal decomposition and photochemistry of (tetrazol-5-yl)acetic acid.

The title compound (tetrazol-5-yl)acetic acid (TA) is an interesting molecule that contains both a carboxylic group and its isostere tetrazolyl group. Out of nine theoretically predicted stable structures of TA, three appeared to be present in solid argon. Thermal decomposition of the species aided by water molecules was studied in detail both experimentally using FTIR matrix isolation technique and theoretically at the B3LYP/6-311++G(2d,2p) level. Experimentally, it was found that the decarboxylation process appeared at the presence of water traces in the system. Theoretically, it was shown that the energy barrier of the water assisted process was lower by ca. 30 kJ mol(-1) comparing to the process without water participation. The UV photolysis of the TA/Ar system was studied using both broad-band and narrow-band sources. The main photoproducts appeared to be carbodiimidylacetic acid and (1H-diaziren-3-yl)acetic acid. The progress of the reactions induced was followed by FTIR spectroscopy, whereas interpretation of the results was supported by quantum chemical calculations (DFT, TD-DFT).

NIR and UV induced transformations of indazole-3-carboxylic acid isolated in low temperature matrices.

The molecular structure and NIR and UV induced phototransformations of indazole-3-carboxylic acid were studied in low temperature argon and nitrogen matrices by FTIR spectroscopy and B3LYP/6-311++G(2d,2p) calculations. Eleven minima of IC were located on the S0 potential energy surface. The three most stable structures were detected experimentally in both matrices after deposition. Upon NIR irradiation a dominant process was rotamerization within the carboxylic group leading to changes in the population of the trans 1HIC1 and cis 1HIC2 forms. In turn, at UV irradiation at λ = 260 nm two new tautomers (2HIC2 and 2HIC3) were generated indicating that the hydrogen atom transfer in pyrazole ring took place. Based on the obtained kinetic curves, differences in the phototransformation rates in different matrices were indicated.

Structure and IR spectroscopic properties of complexes of 1,2,4-triazole and 3-amino-1,2,4-triazole with dinitrogen isolated in solid argon.

Complexes of 1,2,4-triazole (TR) and 3-amino-1,2,4-triazole (AT) with N2 were studied computationally employing MP2 and B3LYPD3 methods and experimentally by FTIR matrix isolation technique. The results show that both triazoles interact specifically with dinitrogen in several different ways. For the 1:1 complexes of 1,2,4-triazole five stable minima were located on the potential energy surface. The most stable of them comprises a weak hydrogen bond formed between the NH group of the ring and the lone pair of the nitrogen molecule. The second most stable structure is bound by the N⋯π bond formed between one of the N atoms of the N2 molecule and the triazole ring. Three other complexes are stabilized by the C-H⋯N and N⋯N van der Waals interactions. In the case of 3-amino-1,2,4-triazole, the two most stable dinitrogen complexes are analogous to those found for the 1,2,4-triazole and involve N-H⋯N and N⋯π bonds. In other structures amino or CH groups act as proton donors to the N2 molecule. The N⋯N van der Waals interactions are also present. The analysis of the infrared spectra of low temperature matrices containing TR or AT and dinitrogen indicates that in both systems mostly 1:1 hydrogen-bonded complexes with the NH group interacting with N2 are present in solid argon.

Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts.

The Candida haemulonii species complex is currently known as C. haemulonii groups I and II. Here we describe C. haemulonii group II as a new species, Candida duobushaemulonii sp. nov., and C. haemulonii var. vulnera as new a variety of C. haemulonii group I using phenotypic and molecular methods. These taxa and other relatives of C. haemulonii (i.e., Candida auris and Candida pseudohaemulonii) cannot be differentiated by the commercial methods now used for yeast identification. Four isolates (C. haemulonii var. vulnera) differed from the other isolates of C. haemulonii in the sequence of the internal transcribed spacer (ITS) regions of the nuclear rRNA gene operon. The new species and the new variety have a multiresistant antifungal profile, which includes high MICs of amphotericin B (geometric mean MIC, 1.18 mg/liter for C. haemulonii var. vulnera and 2 mg/liter for C. duobushaemulonii sp. nov) and cross-resistance to azole compounds. Identification of these species should be based on molecular methods, such as sequence analysis of ITS regions and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Rapid detection of Salmonella sp. by means of a combination of selective enrichment broth and MALDI-TOF MS.

The identification of Salmonella sp. in stool samples usually takes 2 days when employing routine procedures. Fast approaches are necessary in order to shorten the analysis time. The aim of this work was the development of a rapid procedure for the detection of Salmonella sp. from clinical stool samples. Spiked stool samples were cultured in selective selenite enrichment broth. Identifications were directly performed from the liquid broth by the MALDI Biotyper. After the evaluation of this method, the same procedure was applied to clinical samples. Coevally, the samples were streaked on Hektoen agar and single colonies were analyzed by the MALDI Biotyper. For comparison, the liquid broth was plated according to the standard laboratory procedure. A total of 4,847 samples were analyzed for Salmonella sp. In total, 108 Salmonella sp.-positive samples were identified; 66 of these were identified after the streaking of stool samples on Hektoen agar and subsequent MALDI Biotyper analysis of Salmonella sp. suspicious colonies. These and a further 34 samples were detected as Salmonella sp.-positive directly from the selenite enrichment broth on day one. Eight Salmonella sp.-positive samples were not detected before plating of the selenite broth and subsequent MALDI Biotyper analysis on day two. The combination of MALDI Biotyper analysis and selective selenite enrichment broth identification delivers positive results for the majority of the samples already after one day.

Conformational behavior and tautomer selective photochemistry in low temperature matrices: the case of 5-(1H-tetrazol-1-yl)-1,2,4-triazole.

The conformational properties and the photolysis behavior of one of the simplest N-C bonded bicyclic azoles, 5-(1H-tetrazol-1-yl)-1,2,4-triazole (T), were studied in argon and xenon matrices by infrared spectroscopy. Analysis of the experimental results was supported by extensive theoretical calculations carried out at the B3LYP/6-311++G(2d,2p) level of approximation. Out of the eight T minima located on the potential energy surface, the three most stable species were detected in low temperature matrices, namely, 5-(1H-tetrazol-1-yl)-1H-1,2,4-triazole (T1) and two conformers of 5-(1H-tetrazol-1-yl)-2H-1,2,4-triazole (T2a and T2b). With increase of the substrate temperature either during deposition of the matrices or during annealing the T2b → T2a conversion took place, in agreement with the predicted low energy barrier for this transformation (5.38 kJ mol(-1)). Both broad band and narrow band laser UV irradiations of T isolated in Xe and Ar matrices induce unimolecular decomposition involving cleavage of the tetrazole ring of T1 and T2a (T2b) that leads to the production of 1H-1,2,4-triazol-5-yl carbodiimide (P1) and 1H-1,2,4-triazol-3-yl carbodiimide (P2), respectively. When the laser is used, in addition to the main P1 and P2 photoproducts, several minor products could be successfully identified in the matrices: N-cyanocarbodiimide HNCNCN (detected for the first time) associated with nitrilimine HNNCH and HCN. An interesting phenomenon of tautomer-selective photochemistry was observed for the matrix-isolated compound. It could be explained by the different LUMO-HOMO energy gaps estimated for T1, T2a, and T2b, connected with different threshold energies necessary to start the photolysis of T1 and T2a (T2b).

[Complications of venous port systems : Radiological diagnostics and minimally invasive therapy]. / Komplikationen venöser Portsysteme : Radiologische Diagnostik und minimalinvasive Therapie.

INTRODUCTION: Documentation of a correct port placement is a routine investigation in radiology. This article describes typical complications of port catheters and minimally invasive treatment options which can guarantee further use without complications. MATERIAL AND METHODS: From January 2009 to May 2010 a surgical port placement was carried out on 174 patients at the University Clinic in Mannheim and of these, 52 patients were admitted to our institute for radiological imaging of the port. Minimally invasive treatment options are presented based on the observed port complications. RESULTS: Of the 52 patients 7 (13.5%) received a port catheter lysis. A successful port position correction was carried out in 3 (5.8%) patients with a malpositioned port catheter and port removal was recommended in 2 patients (3.8%) due to extensive arm venous thrombosis. A minimally invasive port catheter treatment allowed further use of the port system without operative revision in the corresponding patients. The measures were tolerated very well by the patients without postinterventional complications.

FTIR spectroscopic evidence for new isomers of 3-aminopyrazine-2-carboxylic acid formed in argon matrices upon UV irradiations.

The UV-induced photochemistry and molecular structure of 3-aminopyrazine-2-carboxylic acid were studied in argon matrices by Fourier-transform infrared spectroscopy and B3LYP/6-311++G(2d,2p) calculations. Out of seventeen possible isomers of this molecule located on the singlet potential energy surface the most stable one, APA1 comprising intramolecular O-H···N and N-H···O hydrogen bonds, was detected experimentally in the matrix after deposition. Two new conformers APA2 and APA3 were generated upon irradiation with λ = 280 nm by trans/cis-COOH isomerization and at λ = 360 nm by COOH group rotamerization, respectively, whereas an amino-imino tautomerization leading to IPA1 and IPA2 structures occurred at λ = 305 nm. The reverse reactions were also observed upon irradiation of the matrices at 265, 230 and 400 nm. Simultaneously with the photoisomerizations, a cleavage of the pyrazine ring along with CO2 elimination was observed leading to the formation of carbodiimide and cyanamide derivatives.

Identification of Francisella tularensis by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels.

Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.