An Infection-Relevant Transcriptomic Compendium for Salmonella enterica Serovar Typhimurium

Comparative genomewide expression profiling is a powerful tool in the effort to annotate the mouse genome with biological function. The systematic analysis of RNA expression data of mouse lines from the Munich ENU mutagenesis screen might support the understanding of the molecular biology of such mutants and provide new insights into mammalian gene function. In a direct comparison of DNA microarray experiments of individual versus pooled RNA samples of organs from ENU-induced mouse mutants, we provide evidence that individual RNA samples may outperform pools in some aspects. Genes with high biological variability in their expression levels (noisy genes) are identified as false positives in pooled samples. Evidence suggests that highly stringent housing conditions and standardized procedures for the isolation of organs significantly reduce biological variability in gene expression profiling experiments. Data on wild-type individuals demonstrate the positive effect of controlling variables such as social status, food intake before organ sampling, and stress with regard to reproducibility of gene expression patterns. Analyses of several organs from various ENU-induced mutant lines in general show low numbers of differentially expressed genes. We demonstrate the feasibility to detect transcriptionally affected organs employing RNA expression profiling as a tool for molecular phenotyping.

This work answers the question of whether it is necessary to hybridize individual instead of pooled RNA samples on microarrays for screening gene targets suitable as diagnostic tools for radiation exposure scenarios, while at the same time meeting comparable microarray quality criteria. For developing new clinical diagnostic tools, a two-stage study design was employed in five projects. At first, pooled and not individual RNA samples were hybridized on microarrays for screening purposes. Potential gene candidates were selected based on their fold-change only. This was followed by a validation/quantification step using individual RNA samples and quantitative RT-PCR. Quality criteria from the screening approach with pooled RNA samples were compared with published data from the MicroArray Quality Control (MAQC) consortium that hybridized each reference RNA sample separately and established quality criteria for microarrays. When comparing both approaches, only insignificant differences for quality criteria such as false positives, sensitivity, specificity, and overall agreement were found. However, material, costs, and time were drastically reduced when hybridizing pooled RNA and gene targets applicable for clinical diagnostic purposes could be successfully selected. In search of new diagnostic tools for radiation exposure scenarios, the two stage study design using either pooled or individual RNA samples on microarrays shows comparable quality criteria, but the RNA pooling approach saves unique material, costs, and efforts and successfully selects gene targets that can be used for the desired diagnostic purposes.

<p>Aquaculture products can harbor pathogenic bacteria which are part of the natural microflora. Microbial pathogens associated with fish can be transmitted to human that uses the fishes as source of food or handling. This study aimed to isolate human pathogenic bacteria present in the gills, gut and skin of apparently healthy fishes. Tilapia (<em>Clarias lazera</em>) and <em>Oreochromicniloticus </em>(Cat fish) were collected from Gombe main market and Dadin-kowa of Gombe state. Bacteria were isolated from the fish skin, gill, and gut. Identification of the bacteria was conducted using biochemical tests on specific culture media. Different pathogenic bacteria were isolated from the fish species which appeared yellow or white in colour, small with pin head, while some are circular and irregular that spread all over the media.. The isolates were higher from Dadin-kowa samples compared to Gombe main market. The total bacteria count value ranges between 1.01 x 10<sup>3</sup> to 1.50 x 10<sup>3</sup> for the tilapia fish and 1.04 x 10<sup>3</sup> to 2.20 x 10<sup>3 </sup>cfu/ml for cat fish. The identification results revealed that <em>Staphylococcus aureus</em> and <em>Eshcherichia coli</em> was the most abundant pathogenic bacteria. Presence of <em>Staphylococcus aureus</em> and <em>Eshcherichia coli</em> is attributed to contamination of the fish samples by man through handling and processing. These enteric bacteria from the fishes indicated the organisms faecal contamination and/or water pollution, as well, represents a potential hazard to humans’ life.</p>

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

The Corynebacterium (C.) diphtheriae species complex (CdSC) comprises the closely related pathogenic species C. diphtheriae, C. belfantii, C. rouxii, C. ulcerans, C. silvaticum, C. ramonii and C. pseudotuberculosis according to the taxonomic revisions after 2018. Due to their close relationship, reliable species identification within the CdSC is also challenging using MALDI-TOF mass spectrometry (MS) for fast and reliable identification of bacteria. However, the most commonly used commercial databases for MALDI-TOF MS do not reflect the current CdSC taxonomy. Therefore, the objective of the present study is to expand the Bruker MALDI-Biotyper database version K (MBTK) in order to achieve reliable identification for all currently known Corynebacterium species within the CdSC. Furthermore, the database extensions were verified with a systematic formal validation procedure in accordance with the German guideline for species identification by MALDI- TOF MS. For this, 321 strains including all seven valid species of the CdSC were identified with a set of 328 spectra using the commercial MBTK database and a customer-supplemented modified MBTK. This custom database version included 33 reference spectra of the CdSC and representatives of all four recently described species. The results of the formal validations for both, the commercial and the extended database, combined partly with a single signal analysis, provide a substantiated basis for the identification of bacteria of the CdSC. A key element for a fast up-to-date identification has been the exchange of user-generated sets of MSPs on the open access MALDI-UP catalogue that includes reference spectra for all members of the CdSC. Highlights Using MALDI-TOF MS, all current species of the C. diphtheriae species complex are accurately identified according to the current taxonomy Validation was performed using spectra from different institutions and an approved gold standard protocol The extension and exchange of user created reference spectra via the MALDI User Platform proved to be a key tool for precise identification of bacteria Importance Statement Bacteria belonging to the Corynebacterium (C.) diphtheriae complex are important pathogens for humans and animals. Identification of the species of this complex described since 2018, i.e. C. belfantii, C. rouxii, C. ramonii, and C. silvaticum, using the widely approved MALDI-TOF mass spectrometry is possible. This has been achieved by improved identification using supplemented MALDI reference databases. Comprehensive validation meets the requirements for accurate pathogen identification in accordance with recognised guidelines. This approach of identification is certainly a model for other bacterial species of interest to react on current taxonomic revisions in a short time frame.

Monomorium sp. ant is one of the insects that are considered pests in the hospital environment and potential to be mechanical vectors that can carry and spread pathogenic bacteria and cause nosocomial infections. This study aims to detect and identify pathogenic bacteria found in Monomorium sp. ants originating from the environment of Kendari City Hospital, Southeast Sulawesi. Sampling of Monomorium sp. was carried out by the bait/sugar trap method in 3 locations of Kendari City Hospital, namely internal inpatient rooms, nutrition installations, and laboratory installations. Detection of pathogenic bacteria was carried out by the pour plate method on selective media, namely MacConkey Agar and Mannitol Salt Agar. The identification of bacteria was carried out by numerical-phenetic analysis based on phenotypic characters using the MVSP 3.1 application. The results of the study identified 5 species of pathogenic bacteria found in Monomorium sp. ants from the Kendari City Hospital environment. Three species of bacteria were found in ants from the internal inpatient room, namely Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Brevundimonas vesicularis, and two species found in ants from nutrition installations, namely Pseudomonas luteola and Staphylococcus auricularis. No pathogenic bacteria were found in ants from the laboratory installation.

Introduction: Flies are mechanical vectors that can transmit a variety of pathogenic bacteria, potentially causing human infections, especially in hospital settings. Rapid and accurate identification of these pathogenic bacteria, as well as assessment of their sensitivity to antibiotics, is essential for effective infection control. Methods: Sampling of flies in temporary rubbish bins in the hospital environment. Isolation of pathogenic bacteria from fly body surface by culture method, identification of bacteria using MALDI-TOF Mass Spectrometry technology. Antibiotic sensitivity test was conducted using Viteks 2 Compact to assess the effectiveness of therapy against the isolated bacteria. Ethical approval was obtained from Hasanuddin University Makassar. Results: Four species of pathogenic bacteria, namely Staphylococcus sciuri, Staphylococcus xylosus, Klebsiella pneumoniae, and Proteus mirabilis, were successfully identified quickly and accurately using the MALDI-TOF Mass Spectrometry method. Staphylococcus sciuri and Staphylococcus xylosus are sensitive to Benzylpenicillin, Oxacillin, and Ciprofloxacin, although Staphylococcus sciuri shows moderate resistance to Moxifloxacin. Klebsiella pneumoniae is resistant to Ampicillin, but is effective when combined with Sulbactam, while Proteus mirabilis is generally sensitive, except for Tigecycline. These findings are relevant for infection prevention strategies in hospitals, providing more appropriate antibiotic use guidance and supporting antimicrobial resistance control programs. Conclusion: MALDI-TOF Mass Spectrometry method proved effective in the rapid and accurate identification of pathogenic bacteria from flies. Antibiotic sensitivity testing is essential to determine the most effective therapy and avoid the use of less effective antibiotics.

Chapter 3 - Transcriptomic analysis of physiological systems

Background Endophthalmitis is a serious complication of intraocular surgery.Conventional identification methods for bacteria are becterial culture and smear method, but these laboratory tests spend a long time and have low positive rates.16S rDNA is the bacterial chromosome encoding ribosomal RNA sequences, and it is determined that 16S rDNA sequencing has a high specificity for the identification of bacteria. Objective This study was to identify the infectious bacteria from aqueous humor or vitreous body in the eyes with endophthalmitis by 16S rDNA sequencing technique, and to investigate the diagnosis efficency of 16S rDNA sequencing technique on bacterial endophthalmitis. Methods Anterior chamber fluid (0.1-0.2 ml) or vitreous humor (0.5-1.0 ml) specimens were collected from 5 eyes of 5 patients with endophthalmitis in Qingdao Eye Hospital from June to December 2015 and used for high throughput sequencing, bacterial culture and smear, respectively.Bacteria DNA was extracted from the specimen with D3096-01 trace DNA kit for the amplification of V3-V4 region of 16S rRNA gene and sequencing of hypervariable region of all microbes in the samples by MiSeq Illumina Sequencing Platform.Then the bioinformatic analysis including analysis of taxonomy, abundance and alpha diversity were performed.Nuclease-free water of 50 μl in the centrifuge tube was used as control. Results Five aqueous humor or vitreous body samples were collected, and the positive results were exhibited by smear examination, with the Gram positive bacilli in the trumatic endophthalmitis eye and Gram negative bacilli in the filtering bleb infectious endophthalmitis eye, and all culture results were negative.16S rDNA squencing showed the positive outcomes in all the 5 samples.The high abundent nacteral genuses were Staphylococcus (65.28%), Streptococcus (18.90%) and Pseudomonas (12.76%) in the trumatic endophthalmitis eye; the major components of sample were Pseudomonas (53.68%), Acinetobacter (8.62%) and Limnobacter (5.96%) in the eye with acute endophthalmitis occurring at 2 days following cataract surgery; the major components in the filtering bleb infectious endophthalmitis eye were Moraxella (88.89%) and Pseudomonas (9.52%); the Pseudomonas was major components in the later-onset endophthalmitis eye (84.63%) and the eye with acute endophthalmitis occurring at 1 day after cataract surgery (97.89%). Conclusions A distinct advantage is found in 16S rDNA sequencing technique for the indentification of the pathogenic bacteria in endophthalmitis eyes due to its high positive rate in comparison with bacterial culture and smear method. Key words: Endophthalmitis/microbiology; Aqueous humor/microbiology; Vitreous body/microbiology; DNA, bacterial/analysis; Sequence analysis, DNA; 16S rDNA

This research aimed to identify pathogenic bacteria that infected nile tilapia (Oreochromis niloticus) cultivated at five Districts in Tahuna Regency including Tamako, Kecamatan, Tabukan Tengah, Tabukan Utara, Manganitu and Tahuna Districts, and to analyze water quality (temperature and pH). The research was conducted from July to August 2017. Isolation and identification of bacteria was performed at the Test Laboratory, Station of Fish Guarantee, Quality Control and Fisheries Product Safety Grade II, Tahuna. Target organ examined was kidney. The bacteria was grown on TSA and then isolated. The bacteria was identify by gram stain, motility, aerobic, catalase, oxidase, O/F, glucose and Rimler shots for Aeromonas hydrophila. The bacteria was then identified using Manual for the Identification of Medical Bacteria. From the 30 samples identified, it was found seven pathogenic bacteria infected fish namely A. hydrophila observed at 11 fish (36.6%), Corynebacterium sp. observed at 6 individuals or 20 %, Enterobacteria sp. obtained at 5 fish or 16,6 %, Listeria sp. at 2 fish or 6,6 %, Pseudomonas sp. at 1 fish or 3,3 %, Plesiomonas sp. at 1 fish or 3,3 % and Kurtiha sp. at 1 fish. Water quality parameters at all five districts ranged between 25 and 28° C while pH ranging from 6- 7 and suitable based on the PP N9. 42 Tahun 2001.Keywords: pathogenic bacteria, aquaculture, Oreochromis niloticus

Assessment of cluster analysis of elastic light scatter profiles for the identification of foodborne Bacteria.

The transcription factor Myc has been posited to cause a cell-wide increase in gene expression. But two studies show that Myc, when modulated by other transcription factors, can amplify select targets. See Letters p.483 and p.488 The mammalian Myc oncoprotein is a transcription factor that binds to thousands of promoters. Two current models for Myc function propose that it is either a gene-specific regulator of transcription, or a global amplifier of all active genes. Two groups reporting in this issue of Nature present evidence in support of the idea that Myc regulates specific genes. Arianna Sabo et al. analyse Myc genomic distribution and RNA expression profiles during B-cell lymphomagenesis in mice and Susanne Walz et al. compare normal cells and Myc-transformed tumour cells. Although both groups find that Myc overexpression can result in a general increase in gene expression, the effect is an indirect one. Modulated by various other transcription factors, Myc seems to act primarily by regulating specific groups of genes.

SemiRaman: A self-supervised contrastive representation learning-based framework for semi-supervised Raman spectral identification of pathogenic bacteria.

Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

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 matrix-assisted 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 species-specific 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 Gram-positive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety.