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We present a smartphone-based bacterial colony phenotyping instrument using a reflective elastic light scattering (ELS) pattern and the resolving power of the new instrument. The reflectance-type device can acquire ELS patterns of colonies on highly opaque media as well as optically dense colonies. The novel instrument was built using a smartphone interface and a 532 nm diode laser, and these essential optical components made it a cost-effective and portable device. When a coherent and collimated light source illuminated a bacterial colony, a reflective ELS pattern was created on the screen and captured by the smartphone camera. The collected patterns whose shapes were determined by the colony morphology were then processed and analyzed to extract distinctive features for bacterial identification. For validation purposes, the reflective ELS patterns of five bacteria grown on opaque growth media were measured with the proposed instrument and utilized for the classification. Cross-validation was performed to evaluate the classification, and the result showed an accuracy above 94% for differentiating colonies of E. coli, K. pneumoniae, L. innocua, S. enteritidis, and S. aureus.

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies&rsquo, MinIONTM sequencer and miniPCR bio&rsquo, s miniPCR&trade, thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods, however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

Background To evaluate the accuracy and performance of the Autof MS1000 mass spectrometer in bacteria and yeast identification, 2342 isolates were obtained from microbial cultures of clinical specimens (e.g. blood, cerebrospinal fluid, respiratory tract samples, lumbar puncture fluid, wound samples, stool, and urine) collected in 2019 in Henan Provincial People’s Hospital. Repetitive strains from the same patient were excluded. We tested the Autof MS1000 and Bruker Biotyper mass spectrometry systems and the classical biochemical identification system VITEK 2/API 20C AUX. Inconsistencies in strain identification among the three systems were identified by 16S rDNA and gene sequencing. Results At the species level, the Autof MS1000 and Bruker Biotyper systems had isolate identification accuracies of 98.9 and 98.5%, respectively. At the genus level, the Autof MS1000 and Bruker Biotyper systems were 99.7 and 99.4% accurate, respectively. The instruments did not significantly differ in identification accuracy at either taxonomic level. The frequencies of unreliable identification were 1.1% (26/2342) for the Autof MS1000 and 1.5% (34/2342) for the Bruker Biotyper. In vitro experiments demonstrated that the coincidence rate of the Autof MS1000 mass spectrometer in the identification of five types of bacteria was > 93%, the identification error rate was Conclusions The Autof MS1000 mass spectrometer can be utilised to identify clinical isolates. However, an upgradation of the database is recommended to correctly identify rare strains.

The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.

Bacterial identification is of great importance in clinical diagnosis, environmental monitoring, and food safety control. Among various strategies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has drawn significant interest and has been clinically used. Nevertheless, current bioinformatics solutions use spectral libraries for the identification of bacterial strains. Spectral library generation requires acquisition of MALDI-TOF spectra from monoculture bacterial colonies, which is time-consuming and not possible for many species and strains. We propose a strategy for bacterial typing by MALDI-TOF using protein sequences from public database, that is, UniProt. Ten genes were identified to encode proteins most often observed by MALD-TOF from bacteria through 500 times repeated a 10-fold double cross-validation procedure, using 403 MALDI-TOF spectra corresponding to 14 genera, 81 species, and 403 strains, and the protein sequences of 1276 species in UniProt. The 10 genes were then used to annotate peaks on MALDI-TOF spectra of bacteria for bacterial identification. With the approach, bacteria can be identified at the genus level by searching against a database containing the protein sequences of 42 genera of bacteria from UniProt. Our approach identified 84.1% of the 403 spectra correctly at the genus level. Source code of the algorithm is available at https://github.com/dipcarbon/BacteriaMSLF.

Introduction During the manufacturing of sterile drugs, it is of the utmost importance to meet the minimum requirements for asepsis recommended by the legislations on good manufacturing practices-based efficient environmental monitoring. Aims and methods The availability of relatively simple to use matrix-assisted laser desorption ionization–time of flight mass spectromtomy (MALDI-TOF MS) devices in the last years has changed the laboratory workflows for the microbial identification, mainly in the clinical area. Thus, the objective of this work was to evaluate the suitability of the MALDI-TOF MS technique for the identification of bacteria isolated from the environment of clean rooms used in some stages of the production of a viral vaccine. Eighteen known bacterial species commonly isolated from clean rooms studied were identified by MALDI-TOF technique and by a biochemical technique (BBL Crystal® System). Results Performance of MALDI-TOF MS was better than biochemical technique for correct species identifications (88.89% and 38.89%, respectively) and produced less unreliable identification (5.55% and 22.22%). Conclusion MALDI-TOF MS can be implemented for routine identification of bacteria in a pharmaceutical quality control laboratory, but as a database-dependent system, maybe some isolated not identified by this technique must be additionally studied and, if appropriate, added to an in-house database.

Aims Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. Methods and Results Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1–V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. Conclusions Sequencing the V1–V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. Significance and Impact of the Study We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S~rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although &gt, 99% of reads were correctly classified at the genus level, up to $approx$40% were misclassified at the species level. Both~technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.

Background and Objectives: Bacterial agents are commonly accepted as the main etiology of endodontic infections. A significant proportion of oral bacteria cannot be cultured using existing methods. Since diversity and abundance of bacterial species are different in different populations, the present study was aimed to identify effective microorganisms in persistent endodontic infections in Iranian patients based on culture and molecular biology methods using sequence analysis of 16S rDNA gene. Materials and Methods: Thirty patients with previous failure of endodontic treatment were enrolled in the study. After isolation and disinfection of the tooth surrounding area with 3% sodium hypochlorite and 30% hydrogen peroxide, sampling from the root canals was carried out using two sterile Hedstrom files and two sterile paper points, and then the specimens were transferred to the microbiology laboratory in thioglycolate transport medium so that they undergo aerobic-anaerobic culture, PCR, and 16S rDNA gene sequencing. Results: Of 30 patients (15 women and 15 men), 15 patients had radiographic lesions smaller than 5 mm and other 15 pa- tients had radiographic lesions larger than 5 mm. The mean age of patients was 40.20 ± 13.76 years. A total of 26 patients were asymptomatic. Only four patients had clinical signs such as pain and percussion sensitivity and Tannerella forsythia was the most common bacterium found in this group of patients. 13 bacterial species were found in 11 different genus, one virus strain and one fungus strain. From 30 studied specimens, Enterococcus faecalis was the most common microorganism with prevalence rate of 63.63%. Conclusion: This study showed the type and prevalence of effective bacteria in secondary/persistent endodontic infections in Iranian patients. E. faecalis is the most commonly found microorganism in Iranian patients

Usually, 18-48 h are needed for the identification of microbial pathogens causing urinary tract infections (UTIs) by urine culture. Moreover, antimicrobial susceptibility testing (AST) takes an additional 18-24 h. Rapid identification and AST of the pathogens allow fast and precise treatment. The objective of this study was to shorten the time of diagnosis of UTIs by combining pathogen screening through flow cytometry, microbial identification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS), and AST using the VITEK 2 system for the direct analysis of urine samples. We analyzed 1,638 urine samples from patients with suspected UTIs submitted to the microbiology laboratory for culture. Each urine sample had an approximate volume of 30 mL and was divided into three aliquots. Urine processing included differential centrifugation and two washes to enrich the bacterial fraction for direct MALDI-TOF MS and direct AST. From a total of 1,638 urine samples, 307 were found to be positive through UF-1000i screening. Among them, 265 had significant growth of a single-microorganism. Direct identification was obtained in 229 (86.42%) out of these 265 samples, and no pathogens were misidentified. Moreover, species-level identification was obtained in 163 (88.59%) out of the 184 samples with Gram-negative bacteria, and 27 (38.03%) out of the 71 samples with Gram-positive bacteria. VITEK 2 AST was performed for 117 samples with a single-microorganism. Enterobacteriaceae data showed an agreement rate of antimicrobial categories of 94.83% (1,229/1,296), with minor, major, and very major error rates of 4.17% (54/1,296), 0.92% (12/1,296), and 0.08% (1/1,296), respectively. For Enterococcus spp., the overall categorical agreement was 92.94% (158/170), with a minor error rate of 2.94% (5/170) and major error rate of 4.12% (7/170). The turnaround time of this combined protocol to diagnose UTIs was 1 h for pathogen identification and 6-24 h for AST; noteworthily, only 6-8 h are needed for AST of Enterobacteriaceae using the VITEK 2 system. Overall, our findings show that the combination of flow cytometry, MALDI-TOF MS, and VITEK 2 provided a direct, rapid, and reliable identification and AST method for assessing urine samples, especially for Gram-negative bacterial infections.

The invasion of pathogens into the bloodstream can produce a systemic inflammatory response syndrome that, if not promptly treated, rapidly evolves in acute life-threatening dysfunction of remote organs (septic shock). Current conventional diagnostic tests are microbial blood cultures that need days for results. Therefore, it is crucial the availability of rapid and reliable pathogens detection for prompt diagnosis. In this work, we developed a new method for fast and sensitive detection of bacteria in blood based on the integration of pathogen capture through engineered M13 phages and molecular detection via PCR in miniaturized silicon microchip. Engineered M13 phage-clones exposing specific peptides able to bind Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and a novel identified phage clone, 9IIIB5, for Staphylococcus epidermidis, were used to functionalize magnetic beads and capturing/concentrate the pathogens. Due to the stability of beads-phage-bacteria complex, PCR inhibitors present in the blood were easily removed without detachment of captured bacteria. The complex was used as target for direct amplification into miniaturized silicon chip by a multiplex PCR followed by Real-Time PCR. The procedure was very fast (less than 3 hs) and sensitive (10 cells/reaction) enabling molecular detection inside to the critical period for clinical decision in sepsis. It could be applied to detect pathogens present in very few numbers or in a metabolically inactive state. The results here presented pay the way to future development of portable genetic Point-of-Care in the rapid identification of microorganisms present in the blood stream or also in different matrices, such as waters and foods.

Background A new 23S ribosomal RNA genes-targeted in situ hybridization (ISH) probe to detect global bacterial genomic DNA (59 species from 35 genera; referred to as the GB probe) phagocytized in leukocytes was recently developed. This method provided early and direct evidence of bacterial infection with high sensitivity and specificity in spontaneous bacterial peritonitis ascites. However, the utility of this method in febrile neutropenia (FN) is unknown. Methods We prospectively evaluated the utility of the ISH approach using the GB probe and previously reported probes in patients with neutropenia and fever undergoing chemotherapy at our institution between June 2011 and July 2013. Blood samples for culture analysis and ISH tests were collected simultaneously at the onset of fever; the latter were performed repeatedly. Results Fifty febrile episodes were evaluated. In 24 episodes of fever of unknown origin and 15 episodes of local infection (all negative for blood cultures), ISH tests identified causal bacteria in 21% and 13% of cases, respectively, at the onset of fever. In seven sepsis cases (all positive for blood culture), positive ISH test results at fever onset were achieved in 71%; for two patients with neutrophil counts of 0/μl and 171/μl, respectively, negative results were obtained. Conclusions This new ISH approach could prove useful for early detection of bacteria in patients with neutropenia and blood culture-negative, with fever of unknown etiology after chemotherapy. Using this method in combination with blood culture, even in cases with extremely low neutrophil counts, might contribute to better management of FN.

The recently developed automated, high-throughput monopolar REIMS platform is suited for the identification of clinically important microorganisms. Although already comparable to the previously reported bipolar forceps method, optimization of the geometry of monopolar electrodes, at the heart of the system, holds the most scope for further improvements to be made. For this, sharp tip and round shaped electrodes were optimized to maximize species-level classification accuracy. Following optimization of the distance between the sample contact point and tube inlet with the sharp tip electrodes, the overall cross-validation accuracy improved from 77% to 93% in negative and from 33% to 63% in positive ion detection modes, compared with the original 4 mm distance electrode. As an alternative geometry, round tube shaped electrodes were developed. Geometry optimization of these included hole size, number, and position, which were also required to prevent plate pick-up due to vacuum formation. Additional features, namely a metal “X”-shaped insert and a pin in the middle were included to increase the contact surface with a microbial biomass to maximize aerosol production. Following optimization, cross-validation scores showed improvement in classification accuracy from 77% to 93% in negative and from 33% to 91% in positive ion detection modes. Supervised models were also built, and after the leave 20% out cross-validation, the overall classification accuracy was 98.5% in negative and 99% in positive ion detection modes. This suggests that the new generation of monopolar REIMS electrodes could provide substantially improved species level identification accuracies in both polarity detection modes. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s13361-017-1818-5) contains supplementary material, which is available to authorized users.

Bloodstream infections (BSIs) are among the most concerning bacterial infections. They are one of the leading causes of morbidity and mortality, and occur in 30–70% of critical care patients. The prompt identification of the causative microorganism can help choosing the appropriate antimicrobial therapy that will lead to better clinical outcomes. Blood culture is one of the most relevant tests for microbiological diagnosis of bacterial infections. The introduction of the MALDI-TOF microbiological diagnosis significantly decreased the time of identifying microorganisms. However, it depends on the growth on solid culture medium. In this study, 538 bottles of positive blood cultures were evaluated to test the accuracy of an in house modified protocol. The study sample consisted of 198 Gram-negative and 350 Gram-positive bacteria. In all, 460 (83.94%) species were identified based on the direct plate findings. The protocol allowed the identification of 185/198 (93.43%) of the Gram-negative bacteria, including aerobes, anaerobes, and non-fermenters, and 275/350 (78.85%) of the Gram-positive bacteria. The proposed method has the potential to provide accurate results in comparison to the traditional method with the potential to reduce the turnaround time for the results and optimize antimicrobial therapy in BSI. Keywords: MALDI-TOF, Blood culture, Bacterial identification

Rapid and reliable identification of plant pathogenic bacteria is critical for effective implementation of phytosanitary measures. The genus Erwinia includes a number of economically important plant pathogens such as fire blight agent Erwinia amylovora or Asian pear pathogen Erwinia pyrifoliae, together with closely related plant epiphytes of unknown pathogenicity or even with a potential use for biological control like Erwinia tasmaniensis or Erwinia billingiae, respectively. Current laboratory methods to achieve satisfactory identification and discrimination between species within the Erwinia genus are based on the isolation on semi-selective media, serology, specific PCR and gene locus sequencing: these approaches are complicated and time-consuming, often requiring a priori assumptions over the identity of the isolates. Here we present a streamlined approach based on whole-cell Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) based on the AXIMA mass spectrometer of Shimadzu-Biotech Corp that demonstrates the potential of this technology for quick species identification in plant diagnostics within the genus Erwinia.

Temporary Mediterranean ponds are complex ecosystems which support a high diversity of organisms that include heterotrophic microorganisms, algae, crustaceans, amphibians and higher plants, and have the potential to supply food and a resting place to migratory birds. The role of heterotrophs at the base of the food web in providing energy to the higher trophic levels was studied in temporary ponds in Central and Southern Portugal. The relative quantification of the hetero and autotrophic biomass at the base of the food web in each pond was derived from the polar fatty acid (PLFA) composition of seston through the application of the matrix factorization program CHEMTAX that used specific PLFA and their relative proportion as markers for e.g., classes of bacteria, algae and fungi. The species composition of the culturable microbial communities was identified through their fatty acid profiles. The biomass in the lower trophic level of some ponds presented an even proportion of auto to heterotrophic organisms whilst either bacteria or algae dominated in others. In a selected subset of ponds, the incorporation of bacterial fatty acids was observed to occur in potentially herbivorous zooplankton crustacean. Zooplankton consumed and incorporated bacterial fatty acids into their body tissues, including into their phospholipids, which indicates that energy of heterotrophic origin contributes to the aquatic food webs of temporary ponds.

Traditional bacterial identification methods take one to two days to complete, relying on large bacteria colonies for visual identification. In order to decrease this analysis time in a cost-effective manner, a method to sort and concentrate bacteria based on the bacteria's characteristics itself is needed. One example of such a method is dielectrophoresis, which has been used by researchers to separate bacteria from sample debris and sort bacteria according to species. This work presents variations in which dielectrophoresis can be performed and their associated drawbacks and benefits specifically to bacterial identification. In addition, a potential microfluidic design will be discussed.

Microbiological control is of crucial importance in the pharmaceutical industry regarding the possible bacterial contamination of the environment, water, raw materials and finished products. Molecular identification of bacterial contaminants based on DNA sequencing of the hypervariable 16SrRNA gene has been introduced recently. The aim of this study is to investigate the suitability of gene sequencing using our selection of PCR primers and conditions for rapid and accurate bacterial identification in pharmaceutical industry quality control. DNA was extracted from overnight incubated colonies from 10 bacterial ATCC strains, which are common contaminants in the pharmaceutical industry. A region of bacterial 16SrRNA gene was analyzed by bidirectional DNA sequencing. Bacterial identification based on partial sequencing of the 16SrRNA gene is the appropriate method that could be used in the pharmaceutical industry after adequate validations. We have successfully identified all tested bacteria with more than 99 % similarity to the already published sequences.

15 páginas, 4 tablas, 6 figuras, The rapid identification of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Seafood contaminated with pathogenic bacteria is one of the major causes of food intoxications, and the rapid spoilage of seafood products results in high economic losses. In this study, a collection of the main seafood pathogenic and spoilage Gram-positive bacteria was compiled, including Bacillus spp., Listeria spp., Clostridium spp., Staphylococcus spp. and Carnobacterium spp. The strains, belonging to 20 different species, were obtained from the culture collections and studied by matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A reference library was created, including the spectral fingerprints of 32 reference strains and the extracted peak lists with 10–30 peak masses. Genus-specific as well as speciesspecific peak masses were assigned and could serve as biomarkers for the rapid bacterial identification. Furthermore, the peak mass lists were clustered with the web-application SPECLUST to show the phyloproteomic relationships among the studied strains. Afterwards, the method was successfully applied to identify six strains isolated from seafood by comparison with the reference library. Additionally, phylogenetic analysis based on the 16S rRNA gene was carried out and contrasted with the proteomic approach. This is the first time MALDI-TOF MS fingerprinting is applied to Grampositive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety., This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project AGL2010-19646 from the Spanish Ministry of Science and Technology. The work of K. Bohme and I. C. Fernandez-No is supported by a ‘Maria Barbeito’ and ‘Lucas Labrada’ research contract from Xunta de Galicia.

The major impetus for bacterial identification came after the advent of solid culture media. Morphological appearance of bacterial colonies was often sufficient for their identification in the laboratory. Even in modern times, preliminary identification of most cultivable bacteria is based on such morphological characters. Advances have been made media for the presumptive identifi cation of common organisms encountered in clinical samples. Phenotypic characterisation of bacteria with, physiological tests with a battery of biochemical tests differentiate related bacterial genera as well as confirm their identity. . Each laboratory can select its own method(s) of identification, provided they are based on scientific / epidemiological evidence; clinical laboratory and standards institute (CLSI) is a widely accepted organization and laboratories in many parts of the world follow its recommendations for bacterial identification. Some of the latest advances in identification include Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectroscopy (MALDI-TOF) is a state of art facility used for fast and reliable species-specific identification of bacteria including Mycobacteria and fungi including yeasts. However the single most important factor that decides the method of bacterial identification in any laboratory is the cost involved. In the final analysis, selection of tests for bacterial identification should be based on their standardization with proper scientific basis. Considering the cost and lack of easy availability of commercial kits, we have put forward a simplified and rapid method of identification for most commonly encountered bacterial pathogens causing human infection in India

Hybrid optical gas sensors, based on different organic and inorganic materials, are proposed in this paper, with the aim of using them as optical artificial nose systems. Three types of organic and inorganic dyes, namely zinc-porphyrin, manganese-porphyrin, and zinc-phthalocyanine, were used as gas sensing materials to fabricate a thin-film coating on glass substrates. The performance of the gas sensor was enhanced by a thermal treatment process. The optical absorption spectra and morphological structure of the sensing films were confirmed by UV-Vis spectrophotometer and atomic force microscope, respectively. The optical gas sensors were tested with various volatile compounds, such as acetic acid, acetone, ammonia, ethanol, ethyl acetate, and formaldehyde, which are commonly found to be released during the growth of bacteria. These sensors were used to detect and discriminate between the bacterial odors of three pathogenic species (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) grown in Luria-Bertani medium. Based on a pattern recognition (PARC) technique, we showed that the proposed hybrid optical gas sensors can discriminate among the three pathogenic bacterial odors and that the volatile organic compound (VOC) odor pattern of each bacterium was dependent on the phase of bacterial growth.

The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garçae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi.

Background Bloodstream infections represent serious conditions carrying a high mortality and morbidity rate. Rapid identification of microorganisms and prompt institution of adequate antimicrobial therapy is of utmost importance for a successful outcome. Aiming at the development of a rapid, simplified and efficient protocol, we developed and compared two in-house preparatory methods for the direct identification of bacteria from positive blood culture flasks (BD BACTEC FX system) by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS). Both methods employed saponin and distilled water for erythrocyte lysis. In method A the cellular pellet was overlaid with formic acid on the MALDI TOF target plate for protein extraction, whereas in method B the pellet was exposed to formic acid followed by acetonitrile prior to placing on the target plate. Results Best results were obtained by method A. Direct identification was achieved for 81.9 % and 65.8 % (50.3 % and 26.2 % with scores >2.0) of organisms by method A and method B, respectively. Overall concordance with final identification was 100 % to genus and 97.9 % to species level. By applying a lower cut-off score value, the levels of identification obtained by method A and method B increased to 89.3 % and 77.8 % of organisms (81.9 % and 65.8 % identified with scores >1.7), respectively. Using the lowered score criteria, concordance with final results was obtained for 99.3 % of genus and 96.6 % of species identifications. Conclusion The reliability of results, rapid performance (approximately 25 min) and applicability of in-house method A have contributed to implementation of this robust and cost-effective method in our laboratory. © 2015 Jakovljev and Bergh. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

Introduction. Technological advances during the last years in bacterial identification by molecular biology and mass spectrometry, or Matrix Assisted Laser Desorption lonisation-Time of Flight (MALDI-TOF) have allowed identifying new bacteria involved in urinary tract infections. The purpose of this review is to familiarize clinicians with these bacteria so that they can look at a particular target population and help biologists to suspect and identify them. Method. A bibliographic research was performed using the Medline database concerning the keywords Aerococcus, Actinobaculum, Alloscardovia, Trueperella. Results. If Aerococcus sp. is now well known, Actinobaculum sp., Alloscardovia sp. and Trueperella sp. remain rarely isolated and unknown from clinicians. Importance of these germs could be underestimated because of the identifying and isolation difficulties which existed beforehand. They are particularly found in a population of elderly people with underlying conditions or urological risk factors of urinary tract infection. Actinobaculum schaalii is also found more frequently in young children. Conclusion. Aerococcus sp. et A. schaalii are true uropathogens not to be underestimated. Further studies are still needed to confirm the specific involvement of Trueperella and Alloscardovia in urinary diseases. (C) 2015 Elsevier Masson SAS. All rights reserved.

Identification of emerging bacterial pathogens generally results from a chain of events involving microscopy, serology, molecular tools, and culture. Because of the spectacular molecular techniques developed in the last decades, some authors think that these techniques will shortly supplant culture. The key steps that led to the discovery of emerging bacteria have been reviewed to determine the real contribution of each technique. Historically, microscopy has played a major role. Serology provided indirect evidence for causality. Isolation and culture were crucial, as all emerging bacteria have been grown on artificial media or cell lines or at least propagated in animals. With the use of broad-range polymerase chain reaction, some bacteria have been identified or detected in new clinical syndromes. Culture has irreplaceable advantages for studying emerging bacterial diseases, as it allows antigenic studies, antibiotic susceptibility testing, experimental models, and genetic studies to be carried out, and remains the ultimate goal of pathogen identification.

Rapid evaporative ionization mass spectrometry (REIMS) was investigated for its suitability as a general identification system for bacteria and fungi. Strains of 28 clinically relevant bacterial species were analyzed in negative ion mode, and corresponding data was subjected to unsupervised and supervised multivariate statistical analyses. The created supervised model yielded correct cross-validation results of 95.9%, 97.8%, and 100% on species, genus, and Gram-stain level, respectively. These results were not affected by the resolution of the mass spectral data. Blind identification tests were performed for strains cultured on different culture media and analyzed using different instrumental platforms which led to 97.8–100% correct identification. Seven different Escherichia coli strains were subjected to different culture conditions and were distinguishable with 88% accuracy. In addition, the technique proved suitable to distinguish five pathogenic Candida species with 98.8% accuracy without any further modification to the experimental workflow. These results prove that REIMS is sufficiently specific to serve as a culture condition-independent tool for the identification and characterization of microorganisms.

Universal bacterial primers are often used in PCR-coupled sequencing approaches to investigate environmental and host-associated bacterial communities. Some of these primers can also amplify eukaryotic DNA. This is leading to the submission of datasets to public databases which are erroneously annotated as prokaryotic sequences. The present note sends a message about the risk of submitting incorrectly annotated sequence data and suggests a reliable approach for the sequencing of 16S rRNA genes and identification of bacteria within complex communities.

PyBact is a software written in Python for bacterial identification. The code simulates the predefined behavior of bacterial species by generating a simulated data set based on the frequency table of biochemical tests from diagnostic microbiology textbook. The generated data was used for predictive model construction by machine learning approaches and results indicated that the classifiers could accurately predict its respective bacterial class with accuracy in excess of 99 %.

PyBact is a software written in Python for bacterial identification. The code simulates the predefined behavior of bacterial species by generating a simulated data set based on the frequency table of biochemical tests from diagnostic microbiology textbook. The generated data was used for predictive model construction by machine learning approaches and results indicated that the classifiers could accurately predict its respective bacterial class with accuracy in excess of 99 %., EXCLI Journal ; Vol. 10, 2011

Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained confined to research laboratories. In the last 2 years, the availability of relatively simple to use MALDI-TOF MS devices, which can be utilized in clinical microbiology laboratories, has changed the laboratory workflows for the identification of pathogens. Recently, the first prospective studies regarding the performance in routine bacterial identification showed that MALDI-TOF MS is a fast, reliable and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. For routine bacterial isolates, correct identification by MALDI-TOF MS at the species level was obtained in 84.1–93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%. This review focuses on the current state of use of MALDI-TOF MS for the identification of routine bacterial isolates and on the main difficulties that may lead to erroneous or doubtful identifications.

Mardin ili Dargeçit ve Sırnak ili Güçlükonak ilçeleri sıcak su kaynaklarındansu ve çamur örnekleri alınarak bakteri izolasyonu yapıldı. Bu bakterilerin morfolojik,fizyolojik, biyokimyasal özellikleri, lipit analizi ve 16S rRNA dizi analizi yapılaraktanımlanması yapıldı.Hısta (Güçlükonak) sıcak su kaynagından izole edilen iki tane izolat GE1 veGE2 olarak adlandırıldı. Bu izolatlar çubuk seklinde, gram-pozitif, aerobik, hareketlive spor olusturan termofil karekterde oldukları belirlendi. GE1'in basilleri genellikleikili halde GE2'nin ise tekli halde bulunmaktadır. Üreme sıcaklık aralıgı GE1 için30-65 ºC (optimum 60ºC), GE2 içinde 35-65 ºC (optimum 60ºC) olarak bulundu.Optimum üreme pH'sı GE1'in 9.0, GE2'nin 9.5 olarak bulundu. GE1 ve GE2'ninbüyüme grafigi çizildi ve sırası ile 15. ve 12. saatlerde en yüksek oranda ürediklerigörüldü. GE1 ve GE2'nin nisasta, katalaz, üreaz, fosfataz testleri pozitif tespit edildi.zolatların %1'lik NaCI'e tolerans gösterdikleri saptandı. GE1'in karbon kaynagıolarak galaktoz, sakkaroz, maltoz ve laktozu kullandıgı; glukoz, fruktoz ve gliserolüde zayıf bir sekilde kullandıgı saptandı. GE2'nin maltozu karbon kaynagı olarakkullandıgı, glikoz ve sakkaroz da zayıf olarak kullandıgı tespit edildi.Ilısu (Dargeçit) kaplıcasından izole edilen, AH1 ve AH2 olarak adlandırılanizolatlar çubuk sekilli, gram-pozitif, spor olusturan, aerobik, hareketli termofiliközellikte bakterilerdir. Hücreleri genellikle ikili halde bulunmaktadır. AH1'in üremesıcaklık aralıgı 30-65 ºC (optimumu 60 ºC) olarak bulundu. AH2'nin üreme sıcaklıkaralıgı ise 30-65 ºC (optimum 65 ºC) olarak bulundu. AH1'in pH üreme aralıgı 5.5-10.0 (optimum 7.5) bulundu. AH2'nin pH üreme aralıgı 6.0-11.0 (optimum 7.5)olarak bulundu. AH1 ve AH2' nin büyüme grafigi incelendiginde, çogalmanınsırasıyla 24. saat ve 15. saat oldugu saptandı. zolatların nisasta hidrolizi, katalaz,kazein, lipaz ve fosfataz testleri pozitif olarak tespit edildi. Her iki izolatın da lizozimduyarlılıgı ve sodyum azid duyarlılıgı pozitif görüldü. Ayrıca izolatların %2'likNaCI'e toleranslı oldukları görüldü. AH1'in karbon kaynagı olarak galaktoz vegliserolü zayıf olarak kullandıgı tespit edildi. AH2'nin ise sakkaroz, laktoz vegliserolü zayıf bir sekilde kullandıgı tespit edildi.GE1, GE2, AH1'in lipit analizleri Alicyclobacillus acidocaldarius subsp.rittmannii ve Geobacillus toebii standartları ile yapılan karsılastırmalarda izolatlarınfosfolipitleri, glikolipitleri ve aminolipitlerinin Geobacillus standatlarına benzerlikgösterdigi görüldü.GE1'in 16S rRNA gen dizisinin veri tabanındaki diger türlerlekarsılastırılması soncunda Geobacillus cinsine ait türle yakınlık gösterdigi görüldü.Geobacillus cinsinin yeni bir türü olabilecegi düsünülmektedir.GE2'nin 16S rRNA gen dizi analizi sonuçları diger türlerle karsılastırıldıgındaGeobacillus kaue'a yakınlık gösterdigi görüldü.AH1 ve AH2'nin 16S rRNA gen dizi analizi veri tabanındaki mevcut türlerlekarsılastırıldıgında Anoxybacillus flavithermus'a % 99 oranında yakınlık gösterdigibulunmustur.Anahtar Kelimeler: Bakteri tanımlanması, termofilik bakteri, 16S rRNA, Sıcak sukaynagı In Dargeçit, Mardin and in Güçlükonak, Sirnak districts of hot springs waterand mud samples of bacteria were isolated. Morphological, physiological,biochemical characteristics, lipid analysis and identification 16S rRNA sequenceanalysis of these bacteria was performed and identified.Two strains isolated from hot springs of Hısta (Güçlükonak) was called GE1 andGE2. These isolates were rod shaped, gram-positive, aerobic, and sports animatedcharacter has been identified as forming thermophile. Bacillus of GE1 is usuallyfound as bilateral. Otherwise, bacillus of GE2 is found as asingle. 30-65 ºC (optimum60 °C) for GE1 and 35-65 ºC (optimum 60 °C) for GE2 was found as thetemperature range of reproductive. Optimum growth pH for GE1 and GE2 werefound 9 and 9,5. Growt graphic of GE1 and GE2 were drawn and produce thehighest rate of they were seen in the fifteenth and twelfth hours, respectively. Starch,catalase, urease, phosphatase tests of GE1 and GE2 have determined positive. 1%NaCI tolerance of isolates were found to show. GE1 used galactose, sucrose, maltoseand lactose as carbon source; glucose, fructose and glycerol which are used in a waythat very little was stated. GE2 used maltose as the carbon source, glucose and wereused as the weak.Strains isolated from hot springs of Ilısu (Dargeçit) was named AH1 andAH2. These bacteria are rod-shaped, gram-positive, spore forming, aerobic, activethermophilic properties. Cells of these bacteria are usually bilateral. 30-65 º C(optimum 60 ° C) was found as temperature range of reproductive for AH1, 30-65(optimum 65 ° C) was found as the temperature range of reproductive for AH2.Reproductive pH range was found 5.5-10 (optimum 7.5) for AH1. reproductive pHrange 6-11 (optimum 7.5) was found. Reproductive pH range was found 6-11(optimum 7.5) for AH2. The optimum time to produce for AH1 and AH2 have beenseen 24th and 15th hours, respectively. Starch hydrolysis, catalase, casein, lipase andphosphatase tests of isolates were positive. the lysozyme sensitivity and sodiumazide sensitivity of both isolates have been seen positive. 2 % NaCI tolerance ofisolates were found to show. AH1 as a carbon source used galactose and glycerolpoorly. AH2 also used sucrose, lactose, and glycerol in a weak manner weredetermined.Lipid analysis of GE1, GE2, AH1, the comparison with standard isolatesAlicyclobacillus acidocaldarius subsp. rittmannii and Geobacillus toebii,phospholipids, glycolipids and amino lipids of isolates were similar to the standard ofthe Geobacillus toebii.16S rRNA gene sequence database of GE1 by comparing with other speciesin the genus Geobacillus species were found to sympathize with. A new species ofthe genus Geobacillus is thought to be.16S rRNA gene of GE2 compared with other types of analysis is showedGeobacillus kaue'a sympathize.16S rRNA gene sequence analysis of AH1 and AH2 in comparison with theexisting data base is similar to Anoxybacillus genus and Anoxybacillus flavithermuswas 99 % affinity.Keywords: Bacterial identification, thermophilic bacteria, 16S rRNA, Hot springs 119

O isolamento e a identificação de microrganismos em leite cru se tornam interessantes do ponto de vista de saúde pública, pois dependendo das espécies isoladas, ações direcionadas podem ser tomadas visando a melhoria de sua qualidade. A deterioração do leite é conseqüência sobretudo do crescimento de microrganismos psicrotróficos, que produzem lipases e proteases termoestáveis que não são desnaturadas durante o processo de pasteurização, conferindo sabores e odores rançoso e amargo, respectivamente. Assim, o objetivo deste trabalho foi isolar e identificar os principais gêneros de bactérias pertencentes à família Enterobacteriaceae, Gram-negativas oxidase positiva, gêneros Staphylococcus e Enterococcus, bem como atividade de lipases e proteases de 16 propriedades rurais do município de Boa Esperança-MG. As bactérias Gram-negativas foram isoladas em ágar eosina azul de metileno (EMB) e ágar Entérico Hektoen. Estafilococos foram isolados em ágar Baird-Parker e Enterococcus em ágar KF. Colônias de interesse foram coletadas e submetidas à coloração de Gram, e às provas de catalase e oxidase. Após esses procedimentos, os isolados selecionados foram identificados utilizando-se Bactray I, II e III; Api 20 Strep; e provas sugeridas pelo Bergey's Manual of Determinative Bacteriology. A identificação sorológica de Enterococcus foi realizada utilizando-se Prolex. O leite oriundo das 16 propriedades continha cepas de microrganismos fecais como Escherichia coli e Enterococcus do grupo D de Lancefield. Bactérias Gram-negativas oxidase positiva foram identificadas em cinco propriedades. Staphylococcus foram encontrados em 10 propriedades. O leite coletado nas fazendas investigadas possui microrganismos que comprometem sua qualidade. Todos os grupos de microrganismos testados revelaram atividades de lipase e protease. Isolation and identification of microorganisms in raw milk is interesting from the viewpoint of public health, since, depending on the species isolated, directed actions may be taken to improve milk quality. Milk deterioration is mainly the result of the growth of psychrotrophic microorganisms that produce heat-resistant lipases and proteases which are not denatured during pasteurization, conferring rancid and sour flavors, respectively. Thus, this work aimed at isolating and identifying the main genera of bacteria belonging to the Enterobacteriaceae family, oxidase-positive Gram-negative, Staphylococcus and Enterococcus genera, as well as determining lipase and protease activity in 16 rural farms in Boa Esperança-MG, Brazil. The Gram-negative bacteria were isolated in Eosin Methylene Blue (EMB) and Hektoen Enteric agar media. Staphylococci were isolated in Baird-Parker agar and Enterococcus in KF agar. Colonies of interest were collected and submitted to Gram stain and to catalase and oxidase tests. Following these procedures, the isolates selected were identified using Bactray I, II and III; Api 20 Strep and tests suggested by Bergey's Manual of Determinative Bacteriology. Sorological identification of Enterococcus was carried out using Prolex. Milk from the 16 rural properties contained strains of fecal microorganisms, such as Escherichia coli and Enterococcus from the Lancefield D group. Oxidase-positive Gram-negative bacteria were identified in five farms. Staphylococcus was found in 10 farms. Milk collected from the farms investigated contained microorganisms that compromise its quality. All the microorganism groups tested showed lipase and protease activities.

PyBact is a software written in Python for bacterial identification. The code simulates the predefined behavior of bacterial species by generating a simulated data set based on the frequency table of biochemical tests from diagnostic microbiology textbook. The generated data was used for predictive model construction by machine learning approaches and results indicated that the classifiers could accurately predict its respective bacterial class with accuracy in excess of 99 %.