Conventional Culture-based Methods Research Articles

Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory.

The rapid rise in increasingly resistant bacteria has become a major threat to public health. Antimicrobial susceptibility testing (AST) is crucial in guiding appropriate therapeutic decisions and infection prevention practices for patient care. However, conventional culture-based AST methods are time-consuming and labor-intensive. Therefore, rapid AST approaches exist to address the delayed gap in time to actionable results. There are two main types of rapid AST technologies- phenotypic and genotypic approaches. In this review, we provide a summary of all commercially available rapid AST platforms for use in clinical microbiology laboratories. We describe the technologies utilized, performance characteristics, acceptable specimen types, types of resistance detected, turnaround times, limitations, and clinical outcomes driven by these rapid tests. We also discuss crucial factors to consider for the implementation of rapid AST technologies in a clinical laboratory and what the future of rapid AST holds.

Biocompatible plasma-treated liquids: A sustainable approach for decontaminating gastrointestinal-infection causing pathogens

Nosocomial infections are a serious threat and difficult to cure due to rising antibiotic resistance in pathogens and biofilms. Direct exposure to cold atmospheric plasma (CAP) has been widely employed in numerous biological research endeavors. Nonetheless, plasma-treated liquids (PTLs) formulated with physiological solutions may offer additional benefits such as enhanced portability, and biocompatibility. Additionally, CAP-infused long-lived reactive oxygen and nitrogen species (RONS) such as nitrite (NO2−), nitrate (NO3−), and hydrogen peroxide (H2O2) can synergistically induce their antibacterial activity. Herein, we investigated those argon-plasma jet-treated liquids, including Ringer’s lactate (RL), phosphate-buffered saline (PBS), and physiological saline, have significant antibacterial activity against nosocomial/gastrointestinal-causing pathogens, which might be due to ROS-mediated lipid peroxidation. Combining the conventional culture-based method with propidium iodide monoazide quantitative PCR (PMAxx™-qPCR) indicated that PTLs induce a minimal viable but non-culturable (VBNC) state and moderately affect culturable counts. Specifically, the PTL exposure resulted in pathogenicity dysfunction via controlling T3SS-related effector genes of S. enterica. Overall, this study provides insights into the effectiveness of PTLs for inducing ROS-mediated damage, controlling the virulence of diarrheagenic bacteria, and modulating homeostatic genes.

Next Generation Sequencing in orthopaedic infections – Where is the road headed?

Next-generation sequencing (NGS) has emerged as a game changer in the field of orthopaedic diagnostics, notably in the detection and management of infections associated with prosthetic joints and implants. This paper conducts an exhaustive examination of the pivotal role, outcomes, and prospective future uses of NGS in diagnosing orthopaedic infections. In comparison to conventional culture-based methods, NGS offers a marked improvement in sensitivity thereby facilitating prompt and comprehensive identification of pathogens. This encompasses the ability to detect polymicrobial infections, antibiotic-resistant strains, and previously imperceptible microorganisms. Furthermore, this article delves into the technology's contribution to advancing personalized medicine and promoting judicious antibiotic use. Nonetheless, the seamless integration of NGS into routine clinical practice is impeded by challenges such as substantial financial outlays, the requisite for specialized equipment and expertise, and the intricacy associated with data analysis. Notwithstanding these impediments, the potential for NGS to revolutionize orthopaedic diagnostics remains substantial, with ongoing advancements poised to address current limitations and broaden its scope within clinical applications.

1D ZnO–Au nanocomposites as label-free photoluminescence immunosensors for rapid detection of Listeria monocytogenes

In this study, we explore the potential of 1D ZnO–Au nanocomposites as innovative label-free photoluminescence (PL) immunosensors for rapidly detecting Listeria monocytogenes, a significant concern in food safety. We synthesized ZnO nanorods (ZnO_NR) and nanowires (ZnO_NW), followed by Au deposition to create ZnO_NR/Au and ZnO_NW/Au nanocomposites. Our analyses, including SEM, TEM, Raman spectroscopy, and photoluminescence (PL), revealed distinct structural and optical properties of these nanocomposites, especially noting the superior crystallinity and stability of ZnO_NR/Au. The biosensor performance was evaluated through PL sensitivity to Anti-Listeria antibodies, demonstrating that ZnO_NR with higher concentration of Au nanoparticles exhibited higher sensitivity and a lower limit of detection (LOD), attributed to a greater density of Listeria binding sites. The developed biosensor demonstrated a remarkable limit of detection (LOD) of 8.3 × 102 CFU/mL, rivaling or surpassing conventional culture-based methods and some molecular techniques. This research underscores the critical role of Au deposition duration in optimizing biosensor performance and presents a promising advancement in rapid and sensitive Listeria detection, with significant implications for enhancing food safety protocols.

The Value of Next-Generation Sequencing in Diagnosis and Therapy of Critically Ill Patients with Suspected Bloodstream Infections: A Retrospective Cohort Study.

Bloodstream infection (BSI), a frequent cause of severe sepsis, is a life-threatening complication in critically ill patients and still associated with a high mortality rate. Rapid pathogen identification from blood is crucial for an early diagnosis and the treatment of patients with suspected BSI. For this purpose, novel diagnostic tools on the base of genetic analysis have emerged for clinical application. The aim of this study was to assess the diagnostic value of additional next-generation sequencing (NGS) pathogen test for patients with suspected BSI in a surgical ICU and its potential impact on antimicrobial therapy. In this retrospective single-centre study, clinical data and results from blood culture (BC) and NGS pathogen diagnostics were analysed for ICU patients with suspected BSI. Consecutive changes in antimicrobial therapy and diagnostic procedures were evaluated. Results: 41 cases with simultaneous NGS and BC sampling were assessed. NGS showed a statistically non-significant higher positivity rate than BC (NGS: 58.5% (24/41 samples) vs. BC: 21.9% (9/41); p = 0.056). NGS detected eight different potentially relevant bacterial species, one fungus and six different viruses, whereas BC detected four different bacterial species and one fungus. NGS results affected antimicrobial treatment in 7.3% of cases. Conclusions: NGS-based diagnostics have the potential to offer a higher positivity rate than conventional culture-based methods in patients with suspected BSI. Regarding the high cost, their impact on anti-infective therapy is currently limited. Larger randomized prospective clinical multicentre studies are required to assess the clinical benefit of this novel diagnostic technology.

Molecular characterization of antimicrobial resistance genes of Staphylococcus aureus isolated from mastitic camel milk in Egypt.

Staphylococcus aureus is one of the most common causes of mastitis worldwide. This study aimed to determine the prevalence and antimicrobial resistance (AMR) patterns of S. aureus in mastitic milk samples collected from camel farms in Matrouh Governorate, Egypt. A total of 200 mastitic camel milk samples were evaluated for S. aureus using both conventional culture-based and molecular-based methods. Antibiotic susceptibility testing of S. aureus isolates was conducted using disc diffusion and agar dilution methods, with antibiotic resistance genes identified through polymerase chain reaction with specific primers. Out of samples tested, 60 (30.00%) were positive for S. aureus. The isolates displayed the highest of resistance against piperacillin-tazobactam (55.00%) followed by trimethoprim- sulfamethoxazole (45.00%) and amoxicillin (40.00%). Half of the isolates were multidrug-resistant (MDR). The AMR genes included methicillin-resistant gene (mecA), β-lactamase gene (blaZ), tetracycline resistance gene (tetK), erythromycin resistance gene (ermB) and vancomycin resistant gene (vanA) were detected in 100%, 100%, 95.00%, 90.00% and 20.00% of the isolates, respectively. In conclusion, the presence of MDRS aureus as a cause of clinical camel mastitis is a significant veterinary and public health concern. These findings highlight the importance of proper antibiotic use in Egyptian camel farms and the need for molecular techniques to fully understand the genetic profile of antimicrobial-resistant S. aureus isolates.

FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens.

Rapid and accurate detection of pathogens and antimicrobial-resistant (AMR) genes of the pathogens are crucial for the clinical diagnosis and effective treatment of infectious diseases. However, the time-consuming steps of conventional culture-based methods inhibit the precise and early application of anti-infection therapy. For the prompt treatment of pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle for the ultra-fast detection of antibiotic-resistant pathogens on site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction from a urine sample with FARPA to avoid the usually complicated DNA extraction step. Furthermore, a 36-tube array made from commercial 384-well titre plates was efficiently introduced to perform FARPA in a portable analyser, achieving an increase in the loading sample throughput (from several to several tens), which is quite suitable for the point-of-care testing (POCT) of multiple pathogens and multiple samples. Finally, we tested 92 urine samples to verify the performance of our proposed method. The sensitivities for the detection of E. coli, K. pneumoniae, E. faecium, and E. faecalis were 92.7%, 93.8%, 100% and 88.9%, respectively. The specificities for the detection of the four pathogens were 100%. Consequently, our rapid, low-cost and user-friendly POCT method holds great potential for guiding the rational use of antibiotics and reducing bacterial resistance.

Mitigation of T3SS-mediated virulence in waterborne pathogenic bacteria by multi-electrode cylindrical-DBD plasma-generated nitric oxide water

S. enterica, S. flexneri, and V. parahaemolyticus bacteria are globally recognized to cause severe diarrheal diseases, consisting of Type III Secretion System (T3SS) effectors that help in bacterial infection and virulence in host cells. This study investigates the properties of multi-electrode cylindrical DBD plasma-generated nitric oxide water (MCDBD-PG-NOW) treatment on the survival and virulence of S. enterica, S. flexneri, and V. parahaemolyticus bacteria. The Colony Forming Unit (CFU) assay, live/dead cell staining, lipid peroxidation assay, and bacteria morphological analysis showed substantial growth inhibition of bacteria. Moreover, to confirm the interaction of reactive nitrogen species (RNS) with bacterial membrane biotin switch assay, DAF-FM, and FTIR analysis were carried out, which established the formation of S-nitrosothiols in the cell membrane, intracellular accumulation of RNS, and changes in the cell composition post-PG-NOW treatment. Furthermore, the conventional culture-based method and a quantitative PCR using propidium monoazide showed minimal VBNC induction under similar condition. The efficiency of bacteria to adhere to mammalian colon cells was significantly reduced. In addition, the infection rate was also controlled by disrupting the virulent genes, leading to the collapse of the infection mechanism. This study provides insights into whether RNS generated from PG-NOW might be beneficial for preventing diarrheal infections.

Metagenomics and metatranscriptomics as potential driving forces for the exploration of diversity and functions of micro-eukaryotes in soil.

Micro-eukaryotes are ubiquitous and play vital roles in diverse ecological systems, yet their diversity and functions are scarcely known. This may be due to the limitations of formerly used conventional culture-based methods. Metagenomics and metatranscriptomics are enabling to unravel the genomic, metabolic, and phylogenetic diversity of micro-eukaryotes inhabiting in different ecosystems in a more comprehensive manner. The in-depth study of structural and functional characteristics of micro-eukaryote community residing in soil is crucial for the complete understanding of this major ecosystem. This review provides a deep insight into the methodologies employed under these approaches to study soil micro-eukaryotic organisms. Furthermore, the review describes available computational tools, pipelines, and database sources and their manipulation for the analysis of sequence data of micro-eukaryotic origin. The challenges and limitations of these approaches are also discussed in detail. In addition, this review summarizes the key findings of metagenomic and metatranscriptomic studies on soil micro-eukaryotes. It also highlights the exploitation of these methods to study the structural as well as functional profiles of soil micro-eukaryotic community and to screen functional eukaryotic protein coding genes for biotechnological applications along with the future perspectives in the field.

Neonatal Tissue-derived Extracellular Vesicle Therapy (NEXT): A Potent Strategy for Precision Regenerative Medicine.

Extracellular vesicle (EV)-based therapies have emerged as a promising means in regenerative medicine. However, the conventional EV therapy strategy (using EVs from a single type of cultured cell) displays some limitations, such as inefficient EV production and lack of tissue-specific repair effects. Here, we report that neonatal tissue-derived EV therapy (NEXT) is a potent strategy for precision tissue repair. In brief, large amounts of EVs with higher yield/purity can be readily isolated from desired tissues with less production time/cost compared to the conventional cell culture-based method. Moreover, source factors, such as age and tissue type, can affect the repair efficacy of such tissue-derived EVs in different tissue injury models (skin wounds and acute kidney injury), and neonatal tissue-derived EVs show superior tissue repair potency compared with adult tissue-derived EVs. Different age- or tissue type-derived EVs have distinct composition (e.g., protein) signatures that are likely due to the diverse metabolic patterns of the donor tissues, which may contribute to the specific repair action modes of NEXT in different types of tissue injury. Furthermore, neonatal tissue-derived EVs can be incorporated with bioactive materials (e.g., artificial ECM hydrogel) for advanced tissue repair. This study highlights that our NEXT strategy may provide a new avenue for precision tissue repair in many types of tissue injury. This article is protected by copyright. All rights reserved.

Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp

BackgroundVibrio spp. are a diverse group of ecologically important marine bacteria responsible for several foodborne outbreaks of gastroenteritis around the world. Their detection and characterization are moving away from conventional culture-based methods towards next generation sequencing (NGS)-based approaches. However, genomic methods are relative in nature and suffer from technical biases arising from library preparation and sequencing. Here, we introduce a quantitative NGS-based method that enables the quantitation of Vibrio spp. at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantification via digital PCR (dPCR).ResultsWe developed six DNA standards, called Vibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA libraries via dPCR. To enable Vibrio-Sequin quantification, we validated three duplex dPCR methods to quantify the six targets. LOQs were ranging from 20 to 120 cp/µl for the six standards, whereas the limit of detection (LOD) was ~ 10 cp/µl for all six assays. Subsequently, a quantitative genomics approach was applied to quantify Vibrio-DNA in a pooled DNA mixture derived from several Vibrio species in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR.ConclusionsWe significantly advance existing quantitative (meta)genomic methods by ensuring metrological traceability of NGS-based DNA quantification. Our method represents a useful tool for future metagenomic studies aiming at quantifying microbial DNA in an absolute manner. The inclusion of dPCR into sequencing-based methods supports the development of statistical approaches for the estimation of measurement uncertainties (MU) for NGS, which is still in its infancy.

An Integrated Paper Microfluidic Device Based on Isothermal Amplification for Simple Sample-to-Answer Detection of Campylobacter jejuni.

Campylobacter jejuni is recognized as the most common species in the genus Campylobacter that causes foodborne diseases. The main reservoirs harboring C. jejuni are poultry products, which are associated with most illnesses, creating a demand for effective detection methods to achieve point-of-need diagnostics. We developed an easy-to-use, hybrid paper/polymer-based microfluidic device that integrates paper-based DNA extraction, isothermal nucleic acid amplification, and lateral flow detection. Overall, the recombinase polymerase amplification (RPA) reaction was completed in 20 min and demonstrated 100% specificity to C. jejuni, including 2 reference strains and 6 wild strains isolated from the agroecosystem, 9 other Campylobacter subspecies strains, and 11 non-Campylobacter strains. The limit of detection (LOD) was 46 CFU/mL with DNA extracted on the cellulose paper. The sensitivity was reduced to 460 CFU/mL on the integrated hybrid paper/polymer-based microfluidic device. This device could detect C. jejuni spiked at concentrations ranging from 101 to 102 CFU/g in chicken meat after an enrichment of 5 to 10 h. For C. jejuni levels of >102 CFU/g, it managed to confirm positive results immediately, without bacterial enrichment. RPA reagents and primers remained stable on the paper platform at 22°C for 12 h. After lyophilization and storage on paper, the RPA reaction showed consistent sensitivity for 3 days, and the LOD was reduced to 103 CFU/mL when storage was extended to 25 days. The use of this hybrid paper/polymer-based microfluidic device enabled detection of Campylobacter in foods with high specificity and sensitivity, demonstrating its potential as a reliable point-of-need diagnostic platform for on-site conditions due to its low cost, portability, and simplicity. IMPORTANCE The global health and economic burden of Campylobacter prompts the development of novel detection techniques that can be implemented in resource-limited and on-site settings. This study described point-of-need identification of C. jejuni using a hybrid paper/polymer-based microfluidic device that is easy to operate. This device had high specificity and sensitivity toward C. jejuni and significantly reduced the total analysis time compared to conventional culture-based methods. Nucleic acid extraction was simplified from intensive pipetting to a paper dipstick, making it more convenient for use in the field as a promising tool for future routine surveillance and outbreak investigation.

Rapid Antibiotic Susceptibility Testing of Gram-Negative Bacteria Directly from Urine Samples of UTI Patients Using MALDI-TOF MS.

Urinary tract infections (UTIs) are one of the most common human infections and are most often caused by Gram-negative bacteria such as Escherichia coli. In view of the increasing number of antibiotic-resistant isolates, rapidly initiating effective antibiotic therapy is essential. Therefore, a faster antibiotic susceptibility test (AST) is desirable. The MALDI-TOF MS-based phenotypic antibiotic susceptibility test (MALDI AST) has been used in blood culture diagnostics to rapidly detect antibiotic susceptibility. This study demonstrates for the first time that MALDI AST can be used to rapidly determine antibiotic susceptibility in UTIs directly from patients' urine samples. MALDI-TOF MS enables the rapid identification and AST of Gram-negative UTIs within 4.5 h of receiving urine samples. Six urinary tract infection antibiotics, including ciprofloxacin, cotrimoxazole, fosfomycin, meropenem, cefuroxime, and nitrofurantoin, were analyzed and compared with conventional culture-based AST methods. A total of 105 urine samples from UTI patients contained bacterial isolates for MALDI AST. The combination of ID and AST by MALDI-TOF allowed us to interpret the result according to EUCAST guidelines. An overall agreement of 94.7% was found between MALDI AST and conventional AST for the urinary tract pathogens tested.

Susceptibility of Campylobacter jejuni to Stressors in Agrifood Systems and Induction of a Viable-but-Nonculturable State.

Many bacteria can become viable but nonculturable (VBNC) in response to stressors commonly identified in agrifood systems. Campylobacter is able to enter the VBNC state to evade unfavorable environmental conditions, but how food processing can induce Campylobacter jejuni to enter this state and the potential role of foods in inducing the VBNC state in C. jejuni remains largely unknown. In this study, the culturability and viability of C. jejuni cells were investigated under chlorine treatment (25 ppm), aerobic stress (atmospheric condition), and low-temperature (4°C) conditions that mimicked food processing. In addition, the behaviors of C. jejuni cells in ultrahigh-temperature (UHT) and pasteurized milk were also monitored during refrigerated storage. The numbers of viable and culturable C. jejuni cells in both the pure bacterial culture and food matrices were separately determined by propidium monoazide (PMA)-quantitative PCR (qPCR) and plating assay. The C. jejuni cells lost their culturability but partially retained their viability (1% to 10%) once mixed with chlorine. In comparison, ~10% of C. jejuni cells were induced to enter the VBNC state after 24 h and 20 days under aerobic and low-temperature conditions, respectively. The viability of the C. jejuni cells remained stable during the induction process in UHT (>10%) and pasteurized (>10%) milk. The number of culturable C. jejuni cells decreased quickly in pasteurized milk, but culturable cells could still be detected in the end (day 21). In contrast, the number of culturable C. jejuni cells slowly decreased, and they became undetectable after >42 days in UHT milk. The C. jejuni cells responded differently to various stress conditions and survived in high numbers in the VBNC state in agrifood systems. IMPORTANCE The VBNC state of pathogens can pose risks to food safety and public health because the pathogens cannot be detected using conventional microbiological culture-based methods but can resuscitate under favorable conditions to develop virulence. As a leading cause of human gastroenteritis worldwide, C. jejuni can enter the VBNC state to survive in the environment and food-processing chain with high prevalence. In this study, the effect of food-processing conditions and food products on the development of VBNC state in C. jejuni was investigated, providing a better understanding of the interaction between C. jejuni and the agroecosystem. The knowledge elicited from this study can aid in developing novel intervention strategies to reduce the food safety risks associated with this microbe.

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents

AimsA new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. MethodsSpiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). ResultsNo qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%–100% (69/72–72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. ConclusionsThe developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Rapid detection of Escherichia coli in dairy milk using static headspace-comprehensive two-dimensional gas chromatography.

A new approach is introduced for rapid and reliable bacteria detection in food. Namely, static headspace-comprehensive two-dimensional gas chromatography (HS-GC × GC) with backflushing. The introduced approach provides fast detection of Escherichia coli (E. coli) in enriched ultra-high-temperature processed (UHT) dairy milk. The presence of E. coli may be indicated by detecting microbial volatile organic compounds emanating from test solutions inoculated with E. coli. In the present investigation, HS-GC × GC analysis is preceded by conventional enrichment in nutrient broth and inoculated samples are clearly discernable from controls following as little as 15h sample enrichment. Headspace equilibration for 28min followed by an 8min GC × GC analysis of enriched test solutions reduces time-to-response by approximately one full day compared to conventional culture-based methods. The presence of ethanol, 1-propanol, and acetaldehyde may be used as a putative marker of E. coli contamination in milk and the introduced approach is able to detect single-cell initial bacterial load. Faster, reliable detection of pathogens and/or spoilage microbes in food products is desirable for the food industry. The described approach has great potential to complement the conventional workflow and be utilised for rapid microbial screening of foodstuff.

Oral Microbiome and Dental Caries Development

Dental caries remains the most prevalent oral disease worldwide. The development of dental caries is highly associated with the microbiota in the oral cavity. Microbiological research of dental caries has been conducted for over a century, with conventional culture-based methods and targeted molecular methods being used in order to identify the microorganisms related to dental caries. These methods’ major limitation is that they can identify only part of the culturable microorganisms in the oral cavity. Introducing sequencing-based technology and bioinformatics analysis has boosted oral microbiome research and greatly expanded the understanding of complex oral microbiology. With the continuing revolution of molecular technologies and the accumulated sequence data of the oral microbiome, researchers have realized that microbial composition alone may be insufficient to uncover the relationship between caries and the microbiome. Most updated evidence has coupled metagenomics with transcriptomics and metabolomics techniques in order to comprehensively understand the microbial contribution to dental caries. Therefore, the objective of this article is to give an overview of the research of the oral microbiome and the development of dental caries. This article reviews the classical concepts of the microbiological aspect of dental caries and updates the knowledge of caries microbiology with the results of current studies on the oral microbiome. This paper also provides an update on the caries etiological theory, the microorganisms related to caries development, and the shifts in the microbiome in dental caries development.

Foodborne Pathogens and Antimicrobial Resistance in Ethiopia: An Urgent Call for Action on "One Health".

Foodborne pathogens are the most common cause of foodborne disease worldwide. They contaminate food at all stages of the food chain, at the agricultural production level (soil and irrigation), from animal sources or at the time of preparation by food handlers. Therefore, food security is a critical issue that affects everyone in the world. Current studies indicate that the problem is more severe in low-income countries like Ethiopia. The majority of studies in Ethiopia have been conducted partially on human, animal or environmental samples with conventional culture-based diagnostic methods. Therefore, this review was conducted to gather information on the main foodborne pathogens and identify gaps in their traceability. Clinically important foodborne pathogens in Ethiopia include (A. lubmbricoids, Toxocara spp., H. nana. E. histolytica/dispar, G. intestinalis, H. diminuta and C. belli), bacteria (Salmonella spp, E. coli O157:H7, B. anthracis, Yersinia, C. perfringens, Klebsiella spp. M. paratuberculosis, and L. monocytogenes), and viruses like (rotaviruses, enteroviruses and astroviruses, hepatitis E virus). In Ethiopia, all isolated foodborne bacterial pathogens showed high rates of antimicrobial resistance (AMR). In particular, the most studied foodborne pathogens, Staphylococcus spp., Salmonella spp., and E. coli from specific sources, showing high levels of resistance to most of the antibiotics prescribed in Ethiopia. The occurrence and persistence of AMR in food is one of the main factors causing the spread of antimicrobial resistance in different compartments, humans, animals and the environment. Therefore, strategies of coordination and struggle from a One Health perspective is an urgent strategy to control antibiotic resistance in order to achieve better outcomes for human and animal health.

Deep Learning-Enabled Detection and Classification of Bacterial Colonies Using a Thin-Film Transistor (TFT) Image Sensor

Early detection and identification of pathogenic bacteria such as Escherichia coli (E. coli) is an essential task for public health. The conventional culture-based methods for bacterial colony detection usually take >24 hours to get the final read-out. Here, we demonstrate a bacterial colony-forming-unit (CFU) detection system exploiting a thin-film-transistor (TFT)-based image sensor array that saves ~12 hours compared to the Environmental Protection Agency (EPA)-approved methods. To demonstrate the efficacy of this CFU detection system, a lensfree imaging modality was built using the TFT image sensor with a sample field-of-view of ~10 cm^2. Time-lapse images of bacterial colonies cultured on chromogenic agar plates were automatically collected at 5-minute intervals. Two deep neural networks were used to detect and count the growing colonies and identify their species. When blindly tested with 265 colonies of E. coli and other coliform bacteria (i.e., Citrobacter and Klebsiella pneumoniae), our system reached an average CFU detection rate of 97.3% at 9 hours of incubation and an average recovery rate of 91.6% at ~12 hours. This TFT-based sensor can be applied to various microbiological detection methods. Due to the large scalability, ultra-large field-of-view, and low cost of the TFT-based image sensors, this platform can be integrated with each agar plate to be tested and disposed of after the automated CFU count. The imaging field-of-view of this platform can be cost-effectively increased to >100 cm^2 to provide a massive throughput for CFU detection using, e.g., roll-to-roll manufacturing of TFTs as used in the flexible display industry.