A Comprehensive Review of Innovative Paradigms in Microbial Detection and Antimicrobial Resistance: Beyond Traditional Cultural Methods.

Microbial detection and antimicrobial resistance (AMR) surveillance are critical components of public health efforts to combat infectious diseases and preserve the efficacy of antimicrobial agents. While foundational in microbial identification, traditional cultural methods are often laborious, time-consuming, and limited in their ability to detect AMR markers. In response to these challenges, innovative paradigms have emerged, leveraging advances in molecular biology, genomics, proteomics, nanotechnology, and bioinformatics. This comprehensive review provides an overview of innovative approaches beyond traditional cultural methods for microbial detection and AMR surveillance. Molecular-based techniques such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) offer enhanced sensitivity and specificity, enabling the rapid identification of microbial pathogens and AMR determinants. Mass spectrometry-based methods provide rapid and accurate detection of microbial biomarkers, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and biosensor technologies. Nanotechnology approaches, such as nanoparticle-based assays and nanopore sequencing, offer novel platforms for sensitive and label-free detection of pathogens and AMR markers. Embracing these innovative paradigms holds immense promise for improving disease diagnosis, antibiotic stewardship, and AMR containment efforts. However, challenges such as cost, standardization, and integration with existing healthcare systems must be addressed to realize the full potential of these technologies. By fostering interdisciplinary collaboration and innovation, we can strengthen our ability to detect, monitor, and combat AMR, safeguarding public health for generations.

Characterization and morphological methods for oral biofilm visualization: where are we nowadays?

The oral microbiome represents an essential component of the oral ecosystem whose symbiotic relationship contributes to health maintenance. The biofilm represents a state of living of microorganisms surrounding themselves with a complex and tridimensional organized polymeric support and defense matrix. The substrates where the oral biofilm adhere can suffer from damages due to the microbial community metabolisms. Therefore, microbial biofilm represents the main etiological factor of the two pathologies of dental interest with the highest incidence, such as carious pathology and periodontal pathology. The study, analysis, and understanding of the characteristics of the biofilm, starting from the macroscopic structure up to the microscopic architecture, appear essential. This review examined the morphological methods used through the years to identify species, adhesion mechanisms that contribute to biofilm formation and stability, and how the action of microbicidal molecules is effective against pathological biofilm. Microscopy is the primary technique for the morphological characterization of biofilm. Light microscopy, which includes the stereomicroscope and confocal laser microscopy (CLSM), allows the visualization of microbial communities in their natural state, providing valuable information on the spatial arrangement of different microorganisms within the biofilm and revealing microbial diversity in the biofilm matrix. The stereomicroscope provides a three-dimensional view of the sample, allowing detailed observation of the structure, thickness, morphology, and distribution of the various species in the biofilm while CLSM provides information on its three-dimensional architecture, microbial composition, and dynamic development. Electron microscopy, scanning (SEM) or transmission (TEM), allows the high-resolution investigation of the architecture of the biofilm, analyzing the bacterial population, the extracellular polymeric matrix (EPS), and the mechanisms of the physical and chemical forces that contribute to the adhesion of the biofilm to the substrates, on a nanometric scale. More advanced microscopic methodologies, such as scanning transmission electron microscopy (STEM), high-resolution transmission electron microscopy (HR-TEM), and correlative microscopy, have enabled the evaluation of antibacterial treatments, due to the potential to reveal the efficacy of different molecules in breaking down the biofilm. In conclusion, evidence based on scientific literature shows that established microscopic methods represent the most common tools used to characterize biofilm and its morphology in oral microbiology. Further protocols and studies on the application of advanced microscopic techniques are needed to obtain precise details on the microbiological and pathological aspects of oral biofilm.

Revolutionizing food safety with electrochemical biosensors for rapid and portable pathogen detection.

Foodborne diseases remain a worldwide concern, despite the advances made in sanitation, pathogen surveillance and food safety management systems. The methods routinely applied for detecting pathogens in foods are time consuming, labor intensive and usually require trained and qualified individuals. The objective of this review was to highlight the use of biosensors, with a focus on the electrochemical devices, as promising alternatives for detecting foodborne pathogens. These biosensors present high speed for obtaining results, with the possibility of evaluating foods in real time, at low cost, ease of use, in addition to being compact and portable. These aspects are considered advantageous and suitable for use in food safety management systems. This work also shows some limitations for the application of biosensors, and we present perspectives with the development and use of nanomaterials.

An Alternative Microbiological Validation for an Online Water Bioburden Analyzer.

BACKGROUND: The Mettler-Toledo 7000RMS analyzer is a Bio-Fluorescent Particle Counter (BFPC) used to monitor real-time bioburden results from Purified Water (PW). OBJECTIVE: Validation of the analyzer using 13 microorganisms and a low-intensity, fluorescent, polystyrene bead. METHODS: During the execution of the validation, a laboratory water system that met Purified Water (PW) quality standards was connected to the 7000RMS, and a syringe pump was used to introduce various concentrations of microorganisms and fluorescent polystyrene beads to the analyzer. Samples were collected and tested via the traditional Membrane Filtration (MF) method and the Colony Forming Unit (CFU) plate count results were compared to the Auto-Fluorescent Unit (AFU) of the 7000RMS analyzer. The validation study was designed to follow the guidance in United States Pharmacopeia (USP) Chapter <1223 > (1), European Pharmacopeia (EP) Chapter 5.1.6 (2), PDA Technical Report 33 (3). Concepts and strategies were adapted from EP Chapter 2.6.12 Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests, EP 10.2 (4), European Pharmacopeia Chapter 2.6.1 Sterility, EP 10.2 (5), USP Chapter <61> Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests (6), USP Chapter <71> Sterility Tests (7), Japanese Pharmacopoeia (JP) General Information Chapter G8 Water: Quality Control of Water for Pharmaceutical Use (8). RESULTS AND CONCLUSION: All pre-determined validation acceptance criteria for Accuracy, Specificity, Precision, Limit of Detection (LOD), Limit of Quantitation (LOQ), Linearity, and Range were met. Further, the 7000RMS demonstrated Performance Equivalence to the MF method per USP <1223> but characteristically lacked correlation to the CFU. HIGHLIGHTS: This validation approach highlights the superior capabilities of the 7000RMS when compared against the traditional compendial MF testing method for PW.

Piezoelectric biosensors: shedding light on principles and applications.

The three decades of experience with piezoelectric devices applied in the field of bioanalytical chemistry are shared. After introduction to principles and suitable measuring approaches, active and passive methods based on oscillators and impedance analysis, respectively, the focus is directed towards biosensing approaches. Immunosensing examples are provided, followed by other affinity sensing approaches based on hybridization of nucleic acids, aptamers, monitoring of enzyme activities, and detection of pathogenic microbes. The combination of piezosensors with cell lines and testing of drugs is highlighted, including mechanically active cells. The combination of piezosensors with other measuring techniques providing original hybrid devices is briefly discussed.

An overview of the bacterial microbiome of public transportation systems-risks, detection, and countermeasures.

When we humans travel, our microorganisms come along. These can be harmless but also pathogenic, and are spread by touching surfaces or breathing aerosols in the passenger cabins. As the pandemic with SARS-CoV-2 has shown, those environments display a risk for infection transmission. For a risk reduction, countermeasures such as wearing face masks and distancing were applied in many places, yet had a significant social impact. Nevertheless, the next pandemic will come and additional countermeasures that contribute to the risk reduction are needed to keep commuters safe and reduce the spread of microorganisms and pathogens, but also have as little impact as possible on the daily lives of commuters. This review describes the bacterial microbiome of subways around the world, which is mainly characterized by human-associated genera. We emphasize on healthcare-associated ESKAPE pathogens within public transport, introduce state-of-the art methods to detect common microbes and potential pathogens such as LAMP and next-generation sequencing. Further, we describe and discuss possible countermeasures that could be deployed in public transportation systems, as antimicrobial surfaces or air sterilization using plasma. Commuting in public transport can harbor risks of infection. Improving the safety of travelers can be achieved by effective detection methods, microbial reduction systems, but importantly by hand hygiene and common-sense hygiene guidelines.

Use of a commercial tissue dissociation system to detect Salmonella-contaminated poultry products.

Successful detection of bacterial pathogens in food can be challenging due to the physical and compositional complexity of the matrix. Different mechanical/physical and chemical methods have been developed to separate microorganisms from food matrices to facilitate detection. The present study benchmarked a commercial tissue digestion system that applies both chemical and physical methods to separate microorganisms from tissues against stomaching, a standard process currently utilized by commercial and regulatory food safety laboratories. The impacts of the treatments on the physical properties of the food matrix were characterized along with the compatibility of the methods with downstream microbiological and molecular detection assays. The results indicate the tissue digestion system can significantly reduce the average particle size of the chicken sample relative to processing via a stomacher (P < 0.001) without adversely affecting either real-time PCR (qPCR) or plate counting assays, which are typically used to detect Salmonella. Furthermore, inoculated chicken treated with the GentleMACS resulted in a significant increase (P < 0.003) in the qPCR's detection capabilities relative to stomached controls. Cohen kappa (κ) coefficient and McNemar's test indicate the plating assays and PCR results agree with measurements obtained via the 3 M Molecular Detection System as defined in the MLG standard (κ > 0.62; P > 0.08). Collectively, the results demonstrate that the technique enables detection of pathogens in meat at lower levels of contamination using current industry standard technologies.

Defect-Passivated Photosensor Based on Cesium Lead Bromide (CsPbBr3) Perovskite Quantum Dots for Microbial Detection.

A defect-passivated photosensor based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QD) was fabricated using parylene films, and the photosensor was applied for the microbial detection. The CsPbBr3 perovskite QDs were synthesized to be homogeneous in size under thermodynamic control, and the perovskite QD-based photosensor was fabricated using MoS2 flakes as the electron transfer layer. In this work, a parylene film with functional groups was deposited on a photosensor for physical protection (waterproof) and defect (halide vacancy) passivation of the perovskite QD. As the first effect of the parylene film, the physical protection of the perovskite QD from water was estimated by comparing the photosensor performance after incubation in water. As the second effect of the parylene, the interaction between the functional groups of the parylene film and the halide vacancies of the perovskite QDs was investigated through the bandgap, crystal structure, and trap-state density analysis. Additionally, density functional theory analysis on Mulliken charges, lattice parameters, and Gibbs free energy demonstrated the effect of the defect passivation by parylene films. Finally, the parylene-passivated QD-based photosensor was applied to the detection of two kinds of food-poisoning and gastroduodenal disease bacteria (Listeria monocytogenes and Helicobacter pylori).

Evaluation of TIB Molbiol LightMix® assays for detection of Mycoplasma genitalium and key resistance mutations for macrolides and fluoroquinolones.

The LightMix® Modular Mycoplasma Macrolide and LightMix® Modular parC Fluoroquinolone Resistance assays (TIB Molbiol) were evaluated using sequential Mycoplasma genitalium positive (n = 125) and negative (n = 93) clinical samples. Results were compared to the results of an established commercial assay (ResistancePlus MG assay, SpeeDx Pty Ltd) or Sanger sequencing (for parC). Detection of M. genitalium by the TIB Molbiol assay had a high agreement with the reference assay, with a positive percent agreement (PPA) of 97.6 [95% confidence interval (CI): 93.1-99.5] and negative percent agreement (NPA) of 95.7 (95% CI: 89.5-98.8). From 105 positive samples, macrolide resistance detection had a PPA of 100% (95% CI: 93.7-100) and NPA of 81.3% (95% CI: 67.4-91.1). For the detection of fluroquinolone resistance mutation G248T/S83I or "other mutation" in the quinolone resistance determinant region, from 95 samples there was 100% (95% CI: 86.3-100) sensitivity and 100% (95% CI: 94.5-100) specificity. The understanding of the basis for fluoroquinolone treatment failure is still developing; it is therefore important to use the output of parC-based resistance assays with caution to avoid the inappropriate use of antibiotic therapies, especially considering the limited number of alternative treatments.

Quantitative multiplex polymerase chain reaction in copies ml-1 linearly correlates with standard urine culture in colonies ml-1 for urinary tract infection (UTI) pathogens.

Standard urine culture (SUC) is the current standard method for confirmation of a urinary tract infection (UTI). SUC identifies microorganisms in urine samples and semi-quantifies these as colony-forming units (CFUs) ml-1. In contrast, quantitative multiplex polymerase chain reaction (q-MPCR) is a culture-independent assay in which the microbes are quantified by targeting genomic sequences and reported as cells ml-1, calculated from copies ml-1. Using serial dilutions within the 104-105 cells ml-1 range, the usual reporting range of SUC, this study compared the quantification results based on SUC and q-MPCR for four uropathogens with the control hemocytometer counts. The results revealed a linear relationship and a 1:1 correlation between the q-MPCR and SUC results. Additional q-MPCR quantification of 36 uropathogenic non-fastidious and fastidious bacteria and yeast indicated a reproducible linear correlation in a 1:1 manner with the control counts over a range of cell densities (103-106 cells ml-1). The results confirm that the quantifications by q-MPCR in cells ml-1 and by SUC in CFUs ml-1 are comparable and answer to the lingering question of how the results of these two methods correlate. Moreover, q-MPCR provided accurate quantification of various microorganisms over wider cell density ranges without the time required for microbial growth.

Quantitation of Mycoplasma genitalium using droplet digital PCR.

Mycoplasma genitalium is a sexually transmitted infection with increasing concerns around antimicrobial resistance. Droplet digital PCR (ddPCR) is a rapid quantification method with high precision that may be useful for absolute quantitation of bacteria in samples. This study aimed to develop a ddPCR assay for the quantification of M. genitalium. ddPCR targeting the gene mgpB was established and analysed using the QX100 ddPCR system. The assay was evaluated against quantitated DNA standards, and then in comparison to an established quantitative PCR performed on the Lightcycler 480 II. DNA template of increasing complexity was used, including synthetic double stranded DNA, DNA extracts from laboratory-cultured M. genitalium strains (n = 17) and DNA from M. genitalium-positive clinical samples (n = 21). There was a strong correlation between ddPCR concentration estimates and measured DNA standards (r2 = 0.997), and between ddPCR and qPCR quantitation for different templates (r2 ranging from 0.953 to 0.997). ddPCR reliably detected template in a range from <10 copies per reaction to >104 copies per reaction and demonstrated linearity over dilution series. Concentration estimates by ddPCR were reproducibly less than those determine by qPCR. ddPCR demonstrated precise and reproducible quantitation of M. genitalium with a variety of templates.

Application of Fluorescence In Situ Hybridization (FISH) in Oral Microbial Detection.

Varieties of microorganisms reside in the oral cavity contributing to the occurrence and development of microbes associated with oral diseases; however, the distribution and in situ abundance in the biofilm are still unclear. In order to promote the understanding of the ecosystem of oral microbiota and the diagnosis of oral diseases, it is necessary to monitor and compare the oral microorganisms from different niches of the oral cavity in situ. The fluorescence in situ hybridization (FISH) has proven to be a powerful tool for representing the status of oral microorganisms in the oral cavity. FISH is one of the most routinely used cytochemical techniques for genetic detection, identification, and localization by a fluorescently labeled nucleic acid probe, which can hybridize with targeted nucleic acid sequences. It has the advantages of rapidity, safety, high sensitivity, and specificity. FISH allows the identification and quantification of different oral microorganisms simultaneously. It can also visualize microorganisms by combining with other molecular biology technologies to represent the distribution of each microbial community in the oral biofilm. In this review, we summarized and discussed the development of FISH technology and the application of FISH in oral disease diagnosis and oral ecosystem research, highlighted its advantages in oral microbiology, listed the existing problems, and provided suggestions for future development..

Optimizing Nanopore Sequencing for Rapid Detection of Microbial Species and Antimicrobial Resistance in Patients at Risk of Surgical Site Infections.

Surgical site infections (SSI) are a significant burden to patients and health care systems. We evaluated the use of Nanopore sequencing (NS) to rapidly detect microbial species and antimicrobial resistance (AMR) genes present in intraoperative bile aspirates. Bile aspirates from 42 patients undergoing pancreatic head resection were included. Three methods of DNA extraction using mechanical cell lysis or protease cell lysis were compared to determine the optimum method of DNA extraction. The impact of host DNA depletion, sequence run duration, and use of different AMR gene databases was also assessed. To determine clinical value, NS results were compared to standard culture (SC) results. NS identified microbial species in all culture positive samples. Mechanical lysis improved NS detection of cultured species from 60% to 76%, enabled detection of fungal species, and increased AMR predictions. Host DNA depletion improved detection of streptococcal species and AMR correlation with SC. Selection of AMR database influenced the number of AMR hits and resistance profile of 13 antibiotics. AMR prediction using CARD and ResFinder 4.1 correctly predicted 79% and 81% of the bile antibiogram, respectively. Sequence run duration positively correlated with detection of AMR genes. A minimum of 6 h was required to characterize the biliary microbes, resulting in a turnaround time of 14 h. Rapid identification of microbial species and AMR genes can be achieved by NS. NS results correlated with SC, suggesting that NS may be useful in guiding early antimicrobial therapy postsurgery. IMPORTANCE Surgical site infections (SSI) are a significant burden to patients and health care systems. They increase mortality rates, length of hospital stays, and associated health care costs. To reduce the risk of SSI, surgical patients are administered broad-spectrum antibiotics that are later adapted to target microbial species detected at the site of surgical incision. Use of broad-spectrum antibiotics can be harmful to the patient. We wanted to develop a rapid method of detecting microbial species and their antimicrobial resistance phenotypes. We developed a method of detecting microbial species and predicting resistance phenotypes using Nanopore sequencing. Results generated using Nanopore sequencing were similar to current methods of detection but were obtained in a significantly shorter amount of time. This suggests that Nanopore sequencing could be used to tailor antibiotics in surgical patients and reduce use of broad-spectrum antibiotics.

A Microbial World: Could Metagenomic Next-Generation Sequencing Be Involved in Acute Respiratory Failure?

Background: The usefulness of metagenomic next-generation sequencing (mNGS) in identifying pathogens is being investigated. We aimed to compare the power of microbial identification between mNGS and various methods in patients with acute respiratory failure. Methods: We reviewed 130 patients with respiratory failure, and 184 specimens including blood, bronchoalveolar lavage fluid (BALF), sputum, pleural effusion, ascitic fluid, and urine were tested by mNGS and conventional methods (culture, PCR). We also enrolled 13 patients to evaluate the power of mNGS and pathogen targets NGS (ptNGS) in microbial identifications. Clinical features and microbes detected were analyzed. Results: mNGS outperformed the conventional method in the positive detection rate of Mycobacterium tuberculosis (MTB) (OR, ∞; 95% CI, 1-∞; P < 0.05), bacteria (OR, 3.7; 95% CI, 2.4-5.8; P < 0.0001), fungi (OR, 4.37; 95% CI, 2.7-7.2; P < 0.0001), mycoplasma (OR, 10.5; 95% CI, 31.8-115; P = 0.005), and virus (OR, ∞; 95% CI, 180.7-∞; P < 0.0001). We showed that 20 patients (28 samples) were detected with Pneumocystis jirovecii (P. jirovecii) by mNGS, but not by the conventional method, and most of those patients were immunocompromised. Read numbers of Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), P. jirovecii, cytomegalovirus (CMV), and Herpes simplex virus 1 (HSV1) in BALF were higher than those in other sample types, and the read number of Candida albicans (C. albicans) in blood was higher than that in BALF. We found that orotracheal intubation and type 2 diabetes mellitus (T2DM) were associated with a higher detection rate of bacteria and virus by mNGS, immunosuppression was associated with a higher detection rate of fungi and virus by mNGS, and inflammatory markers were associated with mNGS-positive detection rate of bacteria. In addition, we observed preliminary results of ptNGS. Conclusion: mNGS outperformed the conventional method in the detection of MTB, bacteria, fungi, mycoplasma, and virus. Orotracheal intubation, T2DM, immunosuppression, and inflammatory markers were associated with a higher detection rate of bacteria, fungi, and virus by mNGS. In addition, ptNGS results were consistent with the detection of abundant bacteria, fungi, and mycoplasma in our specimens.

SaQuant: a real-time PCR assay for quantitative assessment of Staphylococcus aureus.

BACKGROUND: Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. RESULTS: In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. CONCLUSIONS: This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

AIEgens for microbial detection and antimicrobial therapy.

Pathogenic microbes can cause infections or diseases in hosts and they pose ongoing threats to human health. Antibiotics have been taken an active role in treating a wide variety of infections or diseases since they were first introduced in the 1940s. However, the emergence of antibiotic-resistant microbes makes these previously effective drugs invalid regrettably. So it is urgently needed to accelerate research and development for new antimicrobial systems and strategies. Recently, luminogens with aggregation-induced emission characteristics (AIEgens) have emerged as powerful fluorescent tools for microbial detection and antimicrobial therapy. In this review, we highlighted the latest advancements of AIEgen-based biofunctional materials and systems in this research field. AIE fluorescent probes have the advantages of excellent sensitivity and rapid response, which make them useful for ultrafast bacterial imaging, bacteria classification, and pathogen discrimination. Early microbial detection and identification could help us study the mechanism of antibiotic resistance more scientifically. Moreover, the AIEgens-based photosensitizers (AIE-PSs) with strong photosensitization show good performance on the efficient elimination of multidrug-resistant bacteria and intracellular bacteria. At the end of the review, a short perspective on aggregate science is concluded.

Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments.

Fluorescence in situ hybridization (FISH) is a canonical tool commonly used in environmental microbiology research to visualize targeted cells. However, the problems of low signal intensity and false-positive signals impede its widespread application. Alternatively, the signal intensity can be amplified by incorporating Hybridization Chain Reaction (HCR) with FISH, while the specificity can be improved through protocol modification and proper counterstaining. Here we optimized the HCR-FISH protocol for studying microbes in environmental samples, particularly marine sediments. Firstly, five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methanococcoides methylutens, and two sets displayed high hybridization efficiency and specificity. Secondly, we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals. Various detachment methods, extraction methods and formulas of hybridization buffer were tested using sediment samples. Thirdly, an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles, providing a reliable reference for FISH imaging. In summary, our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-021-00098-8.

Computational Methods for Strain-Level Microbial Detection in Colony and Metagenome Sequencing Data.

Metagenomic sequencing is a powerful tool for examining the diversity and complexity of microbial communities. Most widely used tools for taxonomic profiling of metagenomic sequence data allow for a species-level overview of the composition. However, individual strains within a species can differ greatly in key genotypic and phenotypic characteristics, such as drug resistance, virulence and growth rate. Therefore, the ability to resolve microbial communities down to the level of individual strains within a species is critical to interpreting metagenomic data for clinical and environmental applications, where identifying a particular strain, or tracking a particular strain across a set of samples, can help aid in clinical diagnosis and treatment, or in characterizing yet unstudied strains across novel environmental locations. Recently published approaches have begun to tackle the problem of resolving strains within a particular species in metagenomic samples. In this review, we present an overview of these new algorithms and their uses, including methods based on assembly reconstruction and methods operating with or without a reference database. While existing metagenomic analysis methods show reasonable performance at the species and higher taxonomic levels, identifying closely related strains within a species presents a bigger challenge, due to the diversity of databases, genetic relatedness, and goals when conducting these analyses. Selection of which metagenomic tool to employ for a specific application should be performed on a case-by case basis as these tools have strengths and weaknesses that affect their performance on specific tasks. A comprehensive benchmark across different use case scenarios is vital to validate performance of these tools on microbial samples. Because strain-level metagenomic analysis is still in its infancy, development of more fine-grained, high-resolution algorithms will continue to be in demand for the future.

Protein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent.

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.