Culture-based Identification Research Articles

Retrospective Comparison of Polymerase Chain Reaction and Culture-Based Identification for Diagnosis of Microsporum canis and Trichophyton Spp. in Shelter Cats and Dogs

Retrospective Comparison of Polymerase Chain Reaction and Culture-Based Identification for Diagnosis of Microsporum canis and Trichophyton Spp. in Shelter Cats and Dogs

Investigating the Role of Microclimate and Microorganisms in the Deterioration of Stone Heritage: The Case of Rupestrian Church from Jac, Romania

Natural stone can undergo disaggregation from various causes, including physical actions such as freeze–thaw cycles, temperature and humidity variations, chemical actions such as the solubilization of minerals by organic and inorganic acids, as well as biological actions due to the colonization of organisms that can produce biocorrosion and biomineralization. This research investigates the impact of microclimatic conditions and microbial activity on the physical and chemical integrity of stone heritage, particularly the biodeterioration caused by fungi in the case of a Romanian rock church. Various analytical techniques were employed, including macroscopic and optical microscopy, Raman spectroscopy, X-ray diffraction, and culture-based identification methods, to characterize the mineral composition and microbial contamination of the rock samples. The analyses revealed that the sandstone consists primarily of quartz (over 90%), muscovite (5–10%), and feldspars. The identified fungi included Cladosporium herbarium, Aspergillus niger, and Mortierella hyalina. The SEM images showed fungal hyphae and spores within the kaolinite–illite matrix, indicating significant microbial colonization and its role in rock deterioration. Additionally, microclimatic data collected over a 12-week period highlighted the substantial fluctuations in temperature and relative humidity within the church, which contribute to the physical and chemical weathering of the stone. This study also noted high levels of particulate matter (PM2.5 and PM10) and volatile organic compounds, which can exacerbate microbial growth and stone decay. The comprehensive analysis underscores the need for targeted preservation strategies that consider both microclimatic factors and microbial colonization to effectively conserve stone heritage sites, ensuring their longevity and structural integrity.

Comparison of Culture-Dependent and Culture-Independent Methods for Routine Identification of Airborne Microorganisms in Speleotherapeutic Caves.

The effective identification of bacterial and fungal isolates is essential for microbiological monitoring in environments like speleotherapeutic caves. This study compares MALDI-TOF MS and the OmniLog ID System, two high-throughput culture-based identification methods. MALDI-TOF MS identified 80.0% of bacterial isolates to the species level, while the OmniLog ID System identified 92.9%. However, species-level matches between the methods were only 48.8%, revealing considerable discrepancies. For discrepant results, MALDI-TOF MS matched molecular identification at the genus level in 90.5% of cases, while the OmniLog ID System matched only in 28.6%, demonstrating MALDI-TOF MS's superiority. The OmniLog ID System had difficulties identifying genera from the order Micrococcales. Fungal identification success with MALDI-TOF MS was 30.6% at the species level, potentially improvable with a customised spectral library, compared to the OmniLog ID System's 16.7%. Metagenomic approaches detected around 100 times more microbial taxa than culture-based methods, highlighting human-associated microorganisms, especially Staphylococcus spp. In addition to Staphylococcus spp. and Micrococcus spp. as indicators of cave anthropisation, metagenomics revealed another indicator, Cutibacterium acnes. This study advocates a multi-method approach combining MALDI-TOF MS, the OmniLog ID System, culture-based, and metagenomic analyses for comprehensive microbial identification. Metagenomic sampling on nitrocellulose filters provided superior read quality and microbial representation over liquid sampling, making it preferable for cave air sample collection.

Performance of 16S rRNA Gene Next-Generation Sequencing and the Culture Method in the Detection of Bacteria in Clinical Specimens.

Effective treatment of infectious diseases requires prompt and accurate bacterial identification and tailored antimicrobial treatments. Traditional culture methods are considered the gold standard, but their effectiveness diminishes for fastidious and hard-to-grow microorganisms. In recent years, molecular diagnostic tools such as 16S rRNA gene next-generation sequencing (16S NGS) have gained popularity in the field. We analysed data from samples submitted for 16S NGS between July 2022 and July 2023 at the Department of Advanced Translational Microbiology in Trieste, Italy. The study included samples submitted for both culture-based identification and 16S NGS. Conventional media were used for culture, and bacterial identification was performed using MALDI-TOF mass spectrometry. The V3 region of the 16S rRNA gene was sequenced using the Ion PGM platform. Among the 123 samples submitted, drainage fluids (38%) and blood (23%) were the most common, with requests predominantly from the Infectious Diseases (31.7%) and Orthopedic (21.13%) Units. In samples collected from patients with confirmed infections, 16S NGS demonstrated diagnostic utility in over 60% of cases, either by confirming culture results in 21% or providing enhanced detection in 40% of instances. Among the 71 patients who had received antibiotic therapies before sampling (mean 2.3 prior antibiotic days), pre-sampling antibiotic consumption did not significantly affect the sensitivity of 16S NGS. In routine microbiology laboratories, combining 16S NGS with culture method enhances the sensitivity of microbiological diagnostics, even when sampling is conducted during antibiotic therapy.

Diagnostic Accuracy of the Abbott SD Bioline MPT64 antigen test for identification of MTB Complex in a U.S. Clinical Mycobacteriology Laboratory

Identification of the Mycobacterium tuberculosis complex (MTBC) from culture and differentiation from other non-tuberculous mycobacterial species is required for rapid diagnosis and accurate treatment. However, gaps exist in culture-based identification of acid-fast bacilli (AFB) positive cultures for rapid rule-out of MTBC in the United States. The SD Bioline™ MPT64 (Abbott Inc, South Korea) lateral flow assay (LFA) has high sensitivity and specificity for the detection of MTBC in liquid culture but has not been evaluated in a clinical mycobacteriology laboratory in the United States. We conducted a diagnostic accuracy study of this LFA for detection of MTBC versus NTMs on AFB positive cultures. A total of 362 tests were performed, with a sensitivity and specificity of 100 % (362/362) across all tests. The SD Bioline MPT64 assay provides accurate test results with AFB-positive liquid cultures and could fill the current gap for rapid rule-out of MTBC in U.S.-based clinical laboratories.

Identification of microorganisms by a rapid PCR panel from positive blood cultures leads to faster optimal antimicrobial therapy – a before-after study

BackgroundThe BioFire® FilmArray® Blood Culture Identification Panel 1 (BF-FA-BCIP) detects microorganisms with high accuracy in positive blood cultures (BC) – a key step in the management of patients with suspected bacteraemia. We aimed to compare the time to optimal antimicrobial therapy (OAT) for the BF-FA-BCIP vs. standard culture-based identification.MethodsIn this retrospective single-centre study with a before-after design, 386 positive BC cases with identification by BF-FA-BCIP were compared to 414 controls with culture-based identification. The primary endpoint was the time from BC sampling to OAT. Secondary endpoints were time to effective therapy, length of stay, (re-)admission to ICU, in-hospital and 30-day mortality. Outcomes were assessed using Cox proportional hazard models and logistic regressions.ResultsBaseline characteristics of included adult inpatients were comparable. Main sources of bacteraemia were urinary tract and intra-abdominal infection (19.2% vs. 22.0% and 16.8% vs. 15.7%, for cases and controls, respectively). Median (95%CI) time to OAT was 25.5 (21.0–31.2) hours with BF-FA-BCIP compared to 45.7 (37.7–51.4) hours with culture-based identification. We observed no significant difference for secondary outcomes.ConclusionsRapid microorganism identification by BF-FA-BCIP was associated with a median 20-h earlier initiation of OAT in patients with positive BC. No impact on length of stay and mortality was noted.Trial registrationClinicaltrials.gov, NCT04156633, registered on November 5, 2019.

Culture-Based Identification of Causative Organisms in Ascitic Fluids of Patients with Spontaneous Bacterial Peritonitis Secondary to Decompensated Liver Disease and their Sensitivities to Ceftriaxone as an Empiric Therapy

OBJECTIVES To identify the pathogens in the ascitic fluids of patients with spontaneous bacterial peritonitis and then to determine their sensitivity pattern to ceftriaxone. METHODOLOGY The cross-sectional study was conducted at the Medical Unit-A, Department of Medicine, Hayatabad Medical Complex, Peshawar, from November 2021 to April 2022. Before ceftriaxone treatment was started, a minimum of 10 ml of ascitic fluid was introduced into a blood culture vial. Only patients with a positive culture were registered, and their information was gathered using a proforma. For statistical analysis, SPSS version 23 was used. RESULTSA total of 96 patients were enrolled in our study. There were 62 (59.52%) male and 34 (40.48%) female patients. Based on the isolation and identification of bacteria, the most prevalent bacteria isolated was Escherichia coli in 36 (37.5%) patients, followed by Acinetobacter Spp in 13 (13.54%) patients, Streptococcus spp in 14 (14.58%), Enterococcus spp in 11 (11.45%), Staphylococcus aureus in 9 (9.39%), MRSA in 8(8.33%) and K. Pneumonia in 5(5.21%) patients. The overall sensitivity of ceftriaxone to gram-positive bacteria was observed in 12 (42.85%) isolates, whereas the overall sensitivity of ceftriaxone to gram-negative bacteria was observed in 25 (36.76%) isolates. (p=0.091) (Figure 6). CONCLUSION Our study concludes that gram-negative bacteria were more prevalent than gram-positive bacteria in ascitic fluids of patients with spontaneous bacterial peritonitis. The most common isolated pathogen was E.coli. Gram-negative was more resistant to ceftriaxone as compared to gram-positive bacteria.

Nano-Biotechnology for Bacteria Identification and Potent Anti-bacterial Properties: A Review of Current State of the Art.

Sepsis is a critical disease caused by the abrupt increase of bacteria in human blood, which subsequently causes a cytokine storm. Early identification of bacteria is critical to treating a patient with proper antibiotics to avoid sepsis. However, conventional culture-based identification takes a long time. Polymerase chain reaction (PCR) is not so successful because of the complexity and similarity in the genome sequence of some bacterial species, making it difficult to design primers and thus less suitable for rapid bacterial identification. To address these issues, several new technologies have been developed. Recent advances in nanotechnology have shown great potential for fast and accurate bacterial identification. The most promising strategy in nanotechnology involves the use of nanoparticles, which has led to the advancement of highly specific and sensitive biosensors capable of detecting and identifying bacteria even at low concentrations in very little time. The primary drawback of conventional antibiotics is the potential for antimicrobial resistance, which can lead to the development of superbacteria, making them difficult to treat. The incorporation of diverse nanomaterials and designs of nanomaterials has been utilized to kill bacteria efficiently. Nanomaterials with distinct physicochemical properties, such as optical and magnetic properties, including plasmonic and magnetic nanoparticles, have been extensively studied for their potential to efficiently kill bacteria. In this review, we are emphasizing the recent advances in nano-biotechnologies for bacterial identification and anti-bacterial properties. The basic principles of new technologies, as well as their future challenges, have been discussed.

Evaluation of the performance of GeneSoC®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/μL, respectively. The detection limit of the MRSA bacterial burden using this system was 104 and 103 CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.

An Interactive, Online Web Map Resource of Global Fusarium oxysporum ff. spp. Diversity and Distribution.

An Interactive, Online Web Map Resource of Global Fusarium oxysporum ff. spp. Diversity and Distribution.

A Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Direct-from-Urine-Specimen Diagnostic for Gram-Negative Pathogens.

Urinary tract infections (UTIs) pose a major public health burden. The vast majority of UTIs are caused by Gram-negative bacteria. Current culture-based pathogen identification methods may require up to 24 to 48 h of incubation. In this study, we developed and evaluated a method for Gram-negative pathogen identification direct from urine, without culture, via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in approximately 1 h. Urine samples were collected (n = 137) from the University of Maryland Medical Center clinical microbiology laboratory. To identify bacteria direct from urine, two methods were evaluated. First, 1 μL of urine was directly spotted onto the MALDI target plate, and second, 1 mL of urine was centrifuged at 8,000 rpm for 5 min before processing using the fast lipid analysis technique (FLAT). Mass spectra were acquired on the Bruker MALDI Biotyper sirius system in the negative-ion mode. Results were compared to those of standard culture methods. When 1 μL of urine was directly spotted, positive agreement was 81.5% (101/124) and, after centrifugation, 94.4% (117/124) relative to that of standard culture methods. Negative agreement for both methods was 100% (13/13). The time to results for both of the specimen preparation methods using the FLAT extraction protocol was approximately 1 h, with minimal hands-on time required (<5 min). The ability to rapidly identify pathogens directly from urine, without the need for culture, allows for faster turnaround times and, potentially, improved patient outcomes. Overall, the FLAT extraction protocol, in combination with lipid A identification, provides a reproducible and accurate method to rapidly identify urinary pathogens. IMPORTANCE This study describes and evaluates a direct-from-urine extraction method that allows identification of Gram-negative bacteria via MALDI-TOF MS within 1 h. Currently, identification of urinary pathogens requires 24 h of culture prior to identification. While this method may not replace culture, we demonstrate its utility in screening for common urinary pathogens. By providing identifications in under 1 h, clinicians can potentially treat patients sooner with more-targeted antimicrobial therapy. In turn, earlier treatment can improve patient outcome and antimicrobial stewardship. Furthermore, MADLI-TOF MS is a readily available, easy-to-use diagnostic tool in clinical laboratories, making implementation of this method possible.

Molecular detection of dermatophytes isolated from tinea corporis infections in the Province of Wasit, Iraq

Background: The detection of fungal components in clinical specimens using direct microscopy in conjunction with culture-based full identification is the basis for dermatophyte identification. Physiological, macro-morphological, and micro-morphological parameters are used to identify colonies phenotypically. In recent years, it has been shown that molecular methods are useful for classifying dermatophyte species. Methods: Fifty samples that were taken from human cases of tinea corporis were examined mycologically. Phenotypic techniques were used to identify isolated dermatophytes. Internal transcribed spacer (ITS) PCR amplification was used for molecular identification. Using a specific primer, PCR was utilized to analyze the 18S rRNA gene in fungal isolates and the genes for virulence factors. Results: Out of Fifty samples with tinea corporis infections identified. Among them, which 3species were identified distribution to 26 cases Trichophyton mentagrophytes with a percentage of 52%, was the most frequently isolated dermatophyte 12 cases Trichophyton rubrum with a percentage 24% and 12 cases Microsporum canis with a percentage 24%. PCR product analysis of the 18S rRNA gene in dermatophytes isolates showed that all dermatophytes ssp. 100% were positive for 18S rRNA gene they obtained dermatophyte isolates (n=50) as follows: 26 Trichophyton mentagrophytes 12 Trichophyton rubrum, 12 Microsporum canis.

Bacterial profile, antimicrobial susceptibility patterns, and associated factors of community-acquired pneumonia among adult patients in Gondar, Northwest Ethiopia: A cross-sectional study.

IntroductionCommunity-acquired pneumonia is associated with higher morbidity, hospitalization, and mortality in adults. Likewise, antimicrobial resistance has increased in recent decades in Ethiopia. Therefore, this study was aimed to determine the bacterial isolates, their antimicrobial susceptibility patterns, and factors associated with community-acquired pneumonia among adult patients in Gondar, Northwest Ethiopia.Materials and methodsThis institutional-based cross-sectional study was conducted from April to June 2021. Sociodemographic, clinical, and other relevant data were collected using a pre-tested questionnaire. A total of 312 sputum specimens were collected using sputum cups and inoculated into blood agar, chocolate agar, mannitol salt agar, and MacConkey agar plates, which were then incubated at 37°C for 24 hours. The bacterial isolates were identified based on Gram staining, colony characteristics, and biochemical tests. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Inducible clindamycin resistance among the S. aureus isolates was detected by the D-test. Data were entered using EPI data version 4.6 and analyzed using SPSS version 20. P-value ≤ 0.05 at 95% CI was considered statistically significant.ResultsOf 312 cases, 39.4% (n = 123; 95% CI: 34.1%–44.9%) were found to have culture-confirmed pneumonia. The most common isolates were K. pneumoniae (31.0%, n = 39), S. pneumoniae (26.2%, n = 33), and S. aureus (20.6%, n = 26). The gram-positive bacteria were susceptible to chloramphenicol (100%) and clindamycin (96.6%). Gram-negative bacteria were susceptible to gentamicin (87.5%), azithromycin (87.1%), ciprofloxacin (86.6%), and ceftriaxone (79.0%) but highly resistant to ampicillin (100%), followed by tetracycline (87.1%), doxycycline (86.4%), co-trimoxazole (80.6%), and amoxicillin-clavulanic acid (79.0%). Overall, 72.2% of the isolates were multi-drug resistant to K. pneumoniae (94.9%, n = 37), E. coli (93.8%, n = 15), and S. pneumoniae (72.7%, n = 24). Only, 7.7% of S. aureus isolates showed inducible clindamycin resistance. Aging (AOR: 3.248, 95% CI: 1.001–10.545, p = 0.050), a history of pneumonia (AOR: 7.004, 95% CI: 3.591–13.658, p = 0.001), alcohol use (AOR: 6.614, 95% CI: 3.399–12.872, p < 0.001), and overcrowded living conditions (AOR: 4.348, 95% CI: 1.964–9.624, p = 0.001) were significantly associated with culture-positive sputum.Conclusion and recommendationsThis study found a high prevalence of bacteria-caused community-acquired pneumonia among adults and low susceptibility to ampicillin, tetracyclines, and amoxicillin-clavulanic acid. Therefore, culture-based bacterial identification and local antibiotic susceptibility testing should be performed regularly. Additionally, new insights into vaccine coverage against highly multi-drug resistant bacteria, particularly K. pneumoniae, are necessary.

Combating Antimicrobial Resistance via Single-Cell Diagnostic Technologies Powered by Droplet Microfluidics.

Antimicrobial resistance is a global threat that if left unchecked could lead to 10 million annual mortalities by 2050. One factor contributing to the rise of multi-drug-resistant (MDR) pathogens is the reliance on traditional culture-based pathogen identification (ID) and antimicrobial susceptibility testing (AST) that typically takes several days. This delay of objective pathogen ID and AST information to inform clinical decision making results in clinicians treating patients empirically often using first-line, broad-spectrum antibiotics, contributing to the misuse/overuse of antibiotics. To combat the rise in MDR pathogens, there is a critical demand for rapid ID and AST technologies. Among the advances in ID and AST technologies in the past decade, single-cell diagnostic technologies powered by droplet microfluidics offer great promise due to their potential for high-sensitivity detection and rapid turnaround time. Our laboratory has been at the forefront of developing such technologies and applying them to diagnosing urinary tract infections (UTIs), one of the most common infections and a frequent reason for the prescription of antimicrobials. For pathogen ID, we first demonstrated the highly sensitive, amplification-free detection of single bacterial cells by confining them in picoliter-scale droplets and detection with fluorogenic peptide nucleic acid (PNA) probes that target their 16S rRNA (rRNA), a well-characterized marker for phylogenic classification. We subsequently improved the PNA probe design and enhanced detection sensitivity. For single-cell AST, we first employed a growth indicator dye and engineered an integrated device that allows us to detect growth from single bacterial cells under antibiotic exposure within 1 h, equivalent to two to three bacterial replications. To expand beyond testing a single antibiotic condition per device, a common limitation for droplet microfluidics, we developed an integrated programmable droplet microfluidic device for scalable single-cell AST. Using the scalable single-cell AST platform, we demonstrated the generation of up to 32 droplet groups in a single device with custom antibiotic titers and the capacity to scale up single-cell AST, and providing reliable pathogen categories beyond a binary call embodies a critical advance. Finally, we developed an integrated ID and AST platform. To this end, we developed a PNA probe panel that can identify nearly 90% of uropathogens and showed the quantitative detection of 16S rRNA from single bacterial cells in droplet-enabled AST after as little as 10 min of antibiotic exposure. This platform achieved both ID and AST from minimally processed urine samples in 30 min, representing one of the fastest turnaround times to date. In addition to tracing the development of our technologies, we compare them with contemporary research advances and offer our perspectives for future development, with the vision that single-cell ID and AST technologies powered by droplet microfluidics can indeed become a useful diagnostic tool for combating antimicrobial resistance.

Carbon Dots as an Emergent Class of Antimicrobial Agents.

Antimicrobial resistance is a recognized global challenge. Tools for bacterial detection can combat antimicrobial resistance by facilitating evidence-based antibiotic prescribing, thus avoiding their overprescription, which contributes to the spread of resistance. Unfortunately, traditional culture-based identification methods take at least a day, while emerging alternatives are limited by high cost and a requirement for skilled operators. Moreover, photodynamic inactivation of bacteria promoted by photosensitisers could be considered as one of the most promising strategies in the fight against multidrug resistance pathogens. In this context, carbon dots (CDs) have been identified as a promising class of photosensitiser nanomaterials for the specific detection and inactivation of different bacterial species. CDs possess exceptional and tuneable chemical and photoelectric properties that make them excellent candidates for antibacterial theranostic applications, such as great chemical stability, high water solubility, low toxicity and excellent biocompatibility. In this review, we will summarize the most recent advances on the use of CDs as antimicrobial agents, including the most commonly used methodologies for CD and CD/composites syntheses and their antibacterial properties in both in vitro and in vivo models developed in the last 3 years.

Adsorption of titanium dioxide nanoparticles onto zebrafish eggs affects colonizing microbiota

Teleost fish embryos are protected by two acellular membranes against particulate pollutants that are present in the water column. These membranes provide an effective barrier preventing particle uptake. In this study, we tested the hypothesis that the adsorption of antimicrobial titanium dioxide nanoparticles onto zebrafish eggs nevertheless harms the developing embryo by disturbing early microbial colonization. Zebrafish eggs were exposed during their first day of development to 2, 5 and 10 mg TiO2 L−1 (NM-105). Additionally, eggs were exposed to gold nanorods to assess the effectiveness of the eggs’ membranes in preventing particle uptake, localizing these particles by way of two-photon microscopy. This confirmed that particles accumulate onto zebrafish eggs, without any detectable amounts of particles crossing the protective membranes. By way of particle-induced X-ray emission analysis, we inferred that the titanium dioxide particles could cover 25–45 % of the zebrafish egg surface, where the concentrations of sorbed titanium correlated positively with concentrations of potassium and correlated negatively with concentrations of silicon. A combination of imaging and culture-based microbial identification techniques revealed that the adsorbed particles exerted antimicrobial effects, but resulted in an overall increase of microbial abundance, without any change in heterotrophic microbial activity, as inferred based on carbon substrate utilization. This effect persisted upon hatching, since larvae from particle-exposed eggs still comprised higher microbial abundance than larvae that hatched from control eggs. Notably, pathogenic aeromonads tolerated the antimicrobial properties of the nanoparticles. Overall, our results show that the adsorption of suspended antimicrobial nanoparticles on aquatic eggs can have cascading effects across different life stages of oviparous animals. Our study furthermore suggests that aggregation dynamics may occur that could facilitate the dispersal of pathogenic bacteria through aquatic ecosystems.

Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections.

Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care.

Translation elongation factor 1-alpha gene as a marker for diagnosing of candidal onychomycosis.

Background and Purpose:Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species.Materials and Methods:For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens.Results:The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively.Conclusion:Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.

657. Evaluation of Nanopore-Based 16S Ribosomal RNA (rRNA) Gene Sequencing for the Development of a Rapid Infection Intervention Clinical Service

BackgroundRapid and accurate identification of bacteria is the basis of appropriate antibiotic treatment and effective clinical decision-making. Next-generation sequencing (NGS) platforms such as Oxford Nanopore Technologies (ONT) holds the promise of a diagnostic revolution by overcoming the limitations of culture-based identification with rapid molecular detection of bacteria. We have developed a pilot to evaluate an ONT 16S rRNA gene assay with the ability to provide real-time analysis and identification of bacterial species. Our aim was to investigate whether long-read sequencing and high-speed analysis can be combined to create a clinically useful, rapid diagnostic tool.MethodsA collection of bacterial isolates representing pathogenic species received by the clinical laboratory over 1 year was assembled. Sample preparation was as described in the ONT 16S protocol and included bead beating sample disruption, MagNA Pure automated nucleic acid extraction (Roche), and PCR amplification (Thermo). Sequencing was performed on the MinION and GridION X5 platforms. Output was analyzed with ONT’s automated EPI2ME 16S pipeline which assigns reads to taxa using BLAST results and the NCBI 16S Bacterial database.ResultsA total of 155 clinical samples with 139 species were sequenced. 119 species were identified at the species level. For 20 samples, a species in the same genus claimed the majority of reads, with the true species being matched to 3%-41% of reads. The average proportion of reads assigned to the correct species was 62.2%, specifically 67% for non-Enterobacteriaceae and 33% for Enterobacteriaceae. 4 clinical samples (3 Bronchoalveolar lavages (BALs), positive for (1) K. pneumoniae, (2) S. pneumoniae, and (3) S. pneumoniae, S. enterica, and S. typhimurium, and 1 bone positive for P. aeruginosa) were also analyzed with sequencing results matching culture.ConclusionEarly results show that 16S rRNA sequencing coupled with real-time analysis was able to accelerate pathogen detection and was able to discriminate the majority of species from a relevant clinical collection. Pipeline refinement is required and subsequent confirmatory consensus-based identification may be a helpful adjunct. Nanopore sequencing shows promise as a rapid bacterial pathogen detection platform for clinical service.Disclosures All authors: No reported disclosures.