Using the Traditional Ex Vivo Whole Blood Model to Discriminate Bacteria by Their Inducible Host Responses.

Whole blood models are rapid and versatile for determining immune responses to inflammatory and infectious stimuli, but they have not been used for bacterial discrimination. Staphylococcus aureus, S. epidermidis and Escherichia coli are the most common causes of invasive disease, and rapid testing strategies utilising host responses remain elusive. Currently, immune responses can only discriminate between bacterial 'domains' (fungi, bacteria and viruses), and very few studies can use immune responses to discriminate bacteria at the species and strain level. Here, whole blood was used to investigate the relationship between host responses and bacterial strains. Results confirmed unique temporal profiles for the 10 parameters studied: IL-6, MIP-1α, MIP-3α, IL-10, resistin, phagocytosis, S100A8, S100A8/A9, C5a and TF3. Pairwise analysis confirmed that IL-6, resistin, phagocytosis, C5a and S100A8/A9 could be used in a discrimination scheme to identify to the strain level. Linear discriminant analysis (LDA) confirmed that (i) IL-6, MIP-3α and TF3 could predict genera with 95% accuracy; (ii) IL-6, phagocytosis, resistin and TF3 could predict species at 90% accuracy and (iii) phagocytosis, S100A8 and IL-10 predicted strain at 40% accuracy. These data are important because they confirm the proof of concept that host biomarker panels could be used to identify bacterial pathogens.

Paper-based fluorescence sensor array with functionalized carbon quantum dots for bacterial discrimination using a machine learning algorithm.

The rapid discrimination of bacteria is currently an emerging trend in the fields of food safety, medical detection, and environmental observation. Traditional methods often require lengthy culturing processes, specialized analytical equipment, and bacterial recognition receptors. In response to this need, we have developed a paper-based fluorescence sensor array platform for identifying different bacteria. The sensor array is based on three unique carbon quantum dots (CQDs) as sensing units, each modified with a different antibiotic (polymyxin B, ampicillin, and gentamicin). These antibiotic-modified CQDs can aggregate on the bacterial surface, triggering aggregation-induced fluorescence quenching. The sensor array exhibits varying fluorescent responses to different bacterial species. To achieve low-cost and portable detection, CQDs were formulated into fluorescent ink and used with an inkjet printer to manufacture paper-based sensor arrays. A smartphone was used to collect the responses generated by the bacteria and platform. Diverse machine learning algorithms were utilized to discriminate bacterial types. Our findings showcase the platform's remarkable capability to differentiate among five bacterial strains, within a detection range spanning from 1.0 × 103 CFU/mL to 1.0 × 107 CFU/mL. Its practicality is further validated through the accurate identification of blind bacterial samples. With its cost-effectiveness, ease of fabrication, and high degree of integration, this platform holds significant promise for on-site detection of diverse bacteria.

A New Theranostic Platform Against Gram-Positive Bacteria Based on Near-Infrared-Emissive Aggregation-Induced Emission Nanoparticles.

Infections induced by Gram-positive bacteria pose a great threat to public health. Antibiotic therapy, as the first chosen strategy against Gram-positive bacteria, is inevitably associated with antibiotic resistance selection. Novel therapeutic strategies for the discrimination and inactivation of Gram-positive bacteria are thus needed. Here, a specific type of aggregation-induced emission luminogen (AIEgen) with near-infrared fluorescence emission as a novel antibiotic-free therapeutic strategy against Gram-positive bacteria is proposed. With the combination of a positively charged group into a highly twisted architecture, self-assembled AIEgens (AIE nanoparticles (NPs)) at a relatively low concentration (5 µm) exhibited specific binding and photothermal effect against living Gram-positive bacteria both in vitro and in vivo. Moreover, toxicity assays demonstrated excellent biocompatibility of AIE NPs at this concentration. All these properties make the AIE NPs as a novel generation of theranostic platform for combating Gram-positive bacteria and highlight their promising potential for in vivo tracing of such bacteria.

Carbon dots for staining bacterial dead cells and distinguishing dead/alive bacteria.

The small molecular dyes such as propidium iodide (PI) always suffer from photo-bleaching and potential toxicity. To tackle the problems, a type of nontoxic carbon dots (CDs) was obtained for dead/alive bacterial distinguishing. This kind of carbon dots has an average size of 1.91 nm and owns carboxyl groups, emerging as excellent candidates for imaging bacterial cells. The negative charges of carboxyl groups lead their avoidance of alive cells while their small size facilitates penetration of dead cells. This kind of nontoxic CDs has effectively differentiated between and alive ones, presenting a highly promising green dye comparing with traditional small molecular dyes.

Detection and discrimination of pathogenic bacteria with nanomaterials-based optical biosensors: A review.

Pathogenic bacteria can pose a great threat to food safety and human health. It is therefore imperative to develop a rapid, portable, and sensitive determination and discrimination method for pathogenic bacteria. Over the past few years, various nanomaterials (NMs) have been employed as desirable nanoprobes because they possess extraordinary properties that can be used for optical signal enabled detection and identification of bacteria. By means of modification, NMs can, depending on different mechanisms, sense targets directly or indirectly, which then provides an essential support for the detection and differentiation of pathogenic bacteria. In this review, recent application of NMs-based optical biosensors for food safety bacterial detection and discrimination is performed, mainly in but not limited to noble metal NMs, fluorescent NMs, and point-of-care testing (POCT). This review also focuses on future trends in bacterial detection and discrimination, and machine learning in performing intelligent rapid detection and multiple accurate identification of bacteria.

Construction of Rhodamine-Based AIE Photosensitizer Hydrogel with Clinical Potential for Selective Ablation of Drug-Resistant Gram-Positive Bacteria In Vivo.

The emergence of powerful antibiotic-resistant bacteria caused by the abuse of antibiotics has become a public health problem. Photodynamic antibacterial therapy is regarded as an innovative and promising antibacterial approach due to its minor side effects and lack of drug resistance. Nevertheless, few photosensitizers (PSs) are reported to have near-infrared (NIR) emission, the ability to rapidly discriminate bacteria, and high photodynamic antibacterial efficiency. In this study, it is reported for the first time that a water-soluble NIR fluorescence emission rhodamine-based photosensitizer with aggregation-inducing emission (AIE) effects, referred to as CS-2I, can efficiently identify and kill Gram-positive bacteria. In a fluorescence imaging experiment with blended bacteria, CS-2I can selectively target Gram-positive bacteria and specifically label Gram-positive bacteria with high efficiency after only 5 min of incubation. Furthermore, CS-2I achieves complete inhibition of methicillin-resistant Staphylococcus aureus (MRSA) at an extremely low concentration (0.5 µm) and light dosage (6 J cm-2 ). Remarkably, CS-2I is mixed with Carbomer 940 to prepare an antibacterial hydrogel dressing (CS-2I@gel), and in vitro and in vivo results demonstrate that CS-2I@gel provides extraordinary performance in photodynamic antibacterial therapy. Hence, this study provides a new strategy and blueprint for the future design of antibacterial materials.

Cationic Tricyclic AIEgens for Concomitant Bacterial Discrimination and Inhibition.

New ionic compounds with aggregation-induced emission (AIE) feature has been widely studied. These AIE-based luminogens (AIEgens) not only effectively resolve aggregation-caused quenching (ACQ) problems that are encountered for most of conventional fluorescent dyes, but also exhibit promising applications in biological imaging, potentially for a wide variety of diseases. However, such an AIE system needs to be further developed. In this work, a series of novel cationic AIEgens that are comprised of tricyclic 2-aminopyridinium derivatives with seven-membered rings are designed and synthesized via a simple, multicomponent reaction. Notably, these AIEgens exhibit the ability to specifically stain gram-positive bacteria. Moreover, a specific AIEgen, BMTAP-7, possesses highly efficient bacteriostatic ability for Staphylococcus aureus (S. aureus) in both liquid medium and solid agar plates, which have a minimum inhibitory concentration (MIC) between 4 and 8 µg mL-1 . Using live-cell imaging and a wash-free process, it is observed that hydrophilic AIEgens are localized to mitochondria, whereas lipophilic AIEgens display specific staining of lysosomes. These AIEgens with bacteriostatic activity hold great promise for distinguishing between bacterial types and inhibiting bacterial infections in situ.

Highly efficient phototheranostics of macrophage-engulfed Gram-positive bacteria using a NIR luminogen with aggregation-induced emission characteristics.

Although phagocytosis serves as the front line to attack invading pathogens, its low bacterial encounter and killing rates leads to an ineffective bactericidal output. In view of this, developing multifunctional theranostic probe to effectively discriminate and ablate intracellular bacteria is highly desirable. However, the shielding effect of the host macrophages put the detection and elimination of macrophage-engulfed bacteria into a challenging task. Herein, we utilize a luminogen with aggregation-induced emission (AIE) characteristics, namely TTVP, as a simple and effective probe for simultaneous tracing and photodynamic killing of intracellular Gram-positive bacteria. With the help of the AIE property, excellent water solubility, near-infrared (NIR) emission and strong reactive oxygen species (ROS) generating ability, TTVP performed ideally to be a targeting agent to intracellular Gram-positive bacteria with high signal contrast, as well as to be a photosensitizer to effectively ablate intracellular bacteria without attacking host macrophages. This work thus provides insights for the next generation antibiosis theranostic application for potential clinical trials.

Multichannel bacterial discrimination based on recognition and disintegration disparity of short antimicrobial peptides.

In this paper, we present a facile and rapid multichannel approach for the simultaneous detection and discrimination of multiple bacterial species. The proposed assay employed four short antimicrobial peptides (SAMPs) for recognition due to their disparity in antibacterial activity against different bacterial strains, and utilized fluorescence measurements to explicate the bacterial recognition and disintegration disparity of the SAMPs. Then, linear discriminant analysis (LDA) was used to effectively discriminate and classify the observed characteristic fluorescence patterns of SAMPs towards target bacteria, exhibiting excellent bacterial discrimination and classification accuracy. This is the first report on the use of SAMPs as recognition units for simultaneous multiple bacterial detection and discrimination. The presented approach was simple, fast, highly repeatable, and required no labelling processes. According to the corresponding LDA discriminant results, six different target bacterial species could be effectively identified and discriminated within 30 min.

Genipin cross-linked carbon dots for antimicrobial, bioimaging and bacterial discrimination.

Multifunctional carbon dots (CDs) present enormous potential in numerous applications and have attracted widespread attention for various applications in the biomedical field. Bacterial infection is a common health issue; the development of antibacterial materials with low toxicity and good biocompatibility is becoming more important. In this work, we synthesized a new type of nitrogen co-doped carbon dots-genipin covalent conjugate (N-CDs-GP) via hydrothermal methods. The microstructure and chemical composition of the N-CDs-GP were characterized. The biocompatibility, stability, antibacterial activity, and fluorescence performance of the N-CDs-GP were assessed. The results revealed that N-CDs-GP possessed high biocompatibility, high light stability, and broad antibacterial activity. Additionally, selective Gram-positive bacterial imaging by N-CDs-GP provided a more rapid method of bacterial detection. The N-CDs-GP have the potential to be applied as bioimaging and antibacterial agents and for bacterial discrimination.

Ultrafast discrimination of Gram-positive bacteria and highly efficient photodynamic antibacterial therapy using near-infrared photosensitizer with aggregation-induced emission characteristics.

With the increase of bacterial infections in clinical practice, it becomes a public health problem which has aroused worldwide attention. Fluorescence imaging-guided photodynamic antibiosis has recently emerged as a promising protocol to solve this problem. However, developing a super powerful fluorescent material allowing facile preparation, long emission wavelength, rapid bacterial discrimination, washing-free staining, and high photodynamic antibacterial efficiency in a single entity, is highly desirable but remains challenging. In this study, we utilize for the first time a water-soluble near-infrared (NIR) emissive luminogen with aggregation-induced emission (AIE) characteristics, namely TTVP, for simultaneous dual applications of Gram-positive bacteria discrimination and photodynamic antibiosis. TTVP is able to selectively target Gram-positive bacteria over Gram-negative bacteria through a washing-free procedure after only 3 s incubation period, which is at least 100-fold shorter than those of previously reported protocols, implying ultrafast bacterial discrimination features. Meanwhile, TTVP exhibits extremely high reactive oxygen species generation efficiency, which is far superior to that of most popularly used photosensitizers, representing one of the best candidates for photodynamic antibiosis. In vitro and in vivo results demonstrate that TTVP provides extraordinary performance on photodynamic antibacterial therapy. This study thus offers a blueprint for the next generation of antibacterial materials.

Phagocyte chase behaviours: discrimination between Gram-negative and Gram-positive bacteria by amoebae.

Phagocytes are cells that pursue, engulf and kill bacteria. They include macrophages and neutrophils of the mammalian immune system, as well as free-living amoebae that hunt and engulf bacteria for food. Phagocytosis can result in diverse outcomes, ranging from sustenance to infection and colonization by either pathogens or beneficial symbionts-and thus, discrimination may be necessary to seek out good bacteria while avoiding bad ones. Here we tested whether the soil amoeba Dictyostelium discoideum can discriminate among different types of bacteria using behavioural assays where amoebae were presented with paired choices of different bacteria. We observed variation in the extent to which the amoebae pursued different types of bacteria, as well as preferential migration towards Gram-negative compared with Gram-positive bacteria. Response profiles were similar for amoebae that originated from different geographical locations, suggesting that chase preference is conserved across much of the species range. While prior work has demonstrated that bacteria use chemotaxis to seek out amoebae they colonize, our work suggests that the opposite also occurs-amoebae can preferentially direct themselves to particular bacteria in the environment. Preferential sensing and response may help to explain why some amoeba-bacteria associations are more common in nature than others.

Antimicrobial peptide hybrid fluorescent protein based sensor array discriminate ten most frequent clinic isolates.

Antimicrobial peptides killed bacteria through intercalating into the bacterial membrane. Their antimicrobial efficiencies varied in bacterial species and were affected by ion strength in the culture medium. A recombinant IGP protein consisted of an antimicrobial peptide, Ib-AMP4 fused with the Green Fluorescent Protein was expressed from E. coli cells and was found to maintain the antimicrobial activity. We demonstrated the interaction between the lipid membranes with IGP by quartz crystal microbalance with dissipation and tried to elucidate the effect of calcium ions by lipopolysaccharide monolayer surface isotherm assays. Ten most frequent clinic isolates were subjected to IGP incubation in buffers containing different calcium ion concentrations. The yielded fluorescent intensities ranging from several thousand to several million, differed greatly between species allowing big coefficient of variances that rendered this method a superior reproducibility and resolution. The classification and data treatment were performed by pattern identification with linear discriminant analysis. Seventy-nine isolates of the 10 most frequent clinic species were classified in the blind test with accuracy >70% by a single measurement and with a 100% accuracy by combined measurements for each species. In conclusion, the concept is based on a solid fact that antimicrobial proteins inhibit bacterial growth at a constant minimal inhibitory concentration through intercalating into the biomembrane. The developed method has a good resolution and high-faulty tolerance rate in discriminating bacteria.

Rapid Screening of Urinary Tract Infection and Discrimination of Gram-Positive and Gram-Negative Bacteria by Automated Flow Cytometric Analysis Using Sysmex UF-5000.

Rapid screening of urinary tract infection is important to determine antibiotic treatment and reduce unnecessary urine culture. We evaluated the performance of the new flow cytometry-based UF-5000 automated urine analyzer (Sysmex, Kobe, Japan). A total of 1,430 urine samples from 1,226 patients were analyzed and compared to urine cultures to which a Previ Isola (bioMérieux, Marcy l'Etoile, France) system was applied. In total, 878 of 1,430 urine cultures (61.4%) produced ≥103 CFU/ml bacterial growth (309 with Gram-negative [GN] bacteria, 517 with Gram-positive [GP] bacteria, and 52 mixed cultures), with 336 samples (23.5%) presenting ≥105 CFU/ml bacterial growth. The ≥105 CFU/ml bacterial growth was detected by a ≥71 bacteria/µl UF-5000 bacterial count with 95% sensitivity and 84% specificity. Using a cutoff of <15 bacteria/µl to determine whether or not to culture, 50.9% of samples were below the cutoff, 94.8 and 99.5% of which presented <104 and <105 CFU/ml of bacterial growth, respectively. The bacterial discrimination performance of the UF-5000 for GN bacteria was superior to that for GP bacteria, and in ≥105 CFU/ml monobacterial samples, the sensitivity and specificity for reporting GN bacteria were 91.7 and 90.0%, respectively. In summary, UF-5000 demonstrated potential utility for the rapid screening of negative bacterial cultures. However, this utility is dependent on the patient population; cutoff optimizations must be performed for specific populations. In addition, UF-5000 presented improved performance in characterizing GP and GN bacteria, although the concurrence rates were not high enough to replace routine cultures.

Aggregation-Induced-Emission Materials with Different Electric Charges as an Artificial Tongue: Design, Construction, and Assembly with Various Pathogenic Bacteria for Effective Bacterial Imaging and Discrimination.

Imaging-based total bacterial count and type identification of bacteria play crucial roles in clinical diagnostics, public health, biological and medical science, and environmental protection. Herein, we designed and synthesized a series of tetraphenylethenes (TPEs) functionalized with one or two aldehyde, carboxylic acid, and quaternary ammonium groups, which were successfully used as fluorescent materials for rapid and efficient staining of eight kinds of representative bacterial species, including pathogenic bacteria Vibrio cholera, Klebsiella pneumoniae, and Listeria monocytogenes and potential bioterrorism agent Yersinia pestis. By comparing the fluorescence intensity changes of the aggregation-induced-emission (AIE) materials before and after bacteria incubation, the sensing mechanisms (electrostatic versus hydrophobic interactions) were simply discussed. Moreover, the designed AIE materials were successfully used as an efficient artificial tongue for bacteria discrimination, and all of the bacteria tested were identified via linear discriminant analysis. Our current work provided a general method for simultaneous broad-spectrum bacterial imaging and species discrimination, which is helpful for bacteria surveillance in many fields.

Discrimination of single-porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy.

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra-species differentiation remains poorly explored. Herein, both transmission Fourier-transform infrared (FTIR) and attenuated total reflectance (ATR)-FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR-spectroscopy techniques is compared with the same collection of unique strains. ATR-FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations.