Bacterial Samples Research Articles

Using image processing and automated classification models to classify microscopic gram stain images

Background and ObjectiveFast and correct classification of bacterial samples are important for accurate diagnostics and treatment. Manual microscopic interpretation of Gram stain samples is both time consuming and operator dependent. The aim of this study was to investigate the potential for developing an automated algorithm for the classification of microscopic Gram stain images. MethodsWe developed and tested two algorithms (using image processing an Casual Probabilistic Network (CPN) and a Random Forest (RF) classification) for the automated classification of Gram stain images. A dataset of 660 images including 33 microbial species (32 bacteria and one fungus) was split into training, validation, and test sets. The algorithms were evaluated based on their ability to correctly classify samples and general characteristics such as aggregation and morphology. ResultsThe CPN correctly classified 633/792 images to achieve an overall accuracy of 80% compared to the RF which correctly classified 782/792 images to achieve an overall accuracy of 99% (p < 0.001). The CPN performed well when distinguishing between GN and GP, with an accuracy of 95% (731/768). The RF also performed well in distinguishing between GN and GP, achieving an accuracy of 99% (767/768) (p < 0.001). ConclusionsThe findings from this study show promising results regarding the potential for an automated algorithm for the classification of microscopic Gram stain images.

Staphyloxanthin inhibitory potential of trans-anethole: A preliminary study

The reduction of staphyloxanthin (STX) production in Staphylococcus aureus under trans-anethole (TA) influence was proven in former studies. However, no tests concerning the impact of TA on a biosynthetic pathway of this carotenoid pigment have been published so far. Thus, for the first time, the present preliminary study evaluated the influence of TA on the expression level of genes (crtOPQMN operon and aldH) encoding STX pathway enzymes. Additional attention was paid to the identification of STX and its intermediates. Gene expression and identification of extracted compounds were conducted using quantitative real-time PCR and HPLC-MS techniques, respectively. The analyzes showed no difference in crtM, crtN, crtO, crtP, crtQ, and aldH gene expression between bacterial samples isolated from the non-stimulated (control) medium and the stimulated one with TA. Compared to the control group that showed the presence of all metabolic intermediates and STX, the TA-treated bacteria were characterized by a lack or a significant reduction of the majority of compounds, except 4,4′-diaponeurosporenoate, the content of which was elevated in the TA-treated sample. Moreover, in silico molecular docking analysis revealed that TA is capable to create relatively strong interactions with both 4,4′-diapophytoene synthase and 4,4′-diapophytoene desaturase. The preliminary findings indicate that the previously observed TA effect reducing the number of S. aureus colonies pigmentation is probably not associated with the expression levels of genes encoding STX pathway enzymes. It has been proven that adding TA to the medium can interfere with the formation of STX at different levels of its biosynthetic pathway.

Ion Addition by Electrolysis to Improve the Quantitative Analysis of Bacteria with MALDI-TOF MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is commonly applied to the identification of bacteria but rarely used for quantitative detection due to the inhomogeneous crystallization of the matrix leading to the unsatisfactory linear relationship between the sample amount and the mass spectrum signals. Herein, we proposed a noninterference ion addition (NIA) method by electrolysis to improve homogeneous crystallization during the evaporation progress of sample droplets on the target plates. The active metal wire was inserted in the droplet as the anode electrode, and metal ions were released through electrolysis. The directional migration of metal ions under the electric field can hinder the migration of matrix molecules to the boundary and homogenize the matrix crystals by forming spherical crystals. Simultaneously, trace cationic surfactant was added to the droplet for pinning the contact surface to define the circle crystallization region. The metal ions from the anode electrode wire were deposited on the surface of the target plates which served as the cathode. Therefore, ion addition has no interference effect on ionization during MALDI-MS detection. This NIA method benefits the homogeneous crystallization and so improves the quantitative analysis. NIA is suitable for biological samples with different matrices, and bacterial samples could be quantitatively analyzed.

Seasonal and spatial variations of bioaerosols and antibiotic resistance bacteria in different wards of the hospital

Introduction: Transmission of bioaerosols through the air is known as an important route for a wide range of nosocomial infections. Therefore, in the present study, we aimed to evaluate the type and diversity of bioaerosols and antibiotic resistance of bacterial bioaerosols in the indoor environments of Sina educational and treatment hospital, Tabriz, Iran. Methods and materials: 150 samples of bacteria and fungi (75 fungi and 75 bacteria) bioaerosol samples were collected on petri dish containing Sabouraud dextrose agar from February to March and June to July 2020 in three periods of daytime (morning, noon and evening) according to National Institute for Occupational Safety and Health (NIOSH 0-800) standard. After sampling, fungal and bacterial samples were incubated and the disk diffusion agar method (Kirby-Bauer) was used for assessing the antibiotic resistance. Results: The concentration of bioaerosols varied significantly in different wards. In addition, the concentration of bioaerosols in winter was observed to be higher than in summer. The highest and lowest airborne fungal concentrations were found in burns operating room and men's infectious ward (49 CFU/m3) and children's burns ward (28 CFU/m3), respectively. The predominantly isolated bacteria were Streptococcus spp. (38%) and Staphylococcus spp. (37%). Also, the main isolated fungi belonged to the genera Aspergillus (75.9%) and Penicillium (22.5%). The highest rates of antibiotic resistance were observed for colistin (100%) in Gram-negative and penicillin (84.2%) in Gram-positive. Conclusion: Timely and regular disinfection of hospital wards can affect the density of bioaerosols. Owing to the prevalence of COVID-19 epidemic in the world, the staff and patients often were wearing masks, gloves and special clothing as well as using disinfectants to prevent coronavirus infection in wards during the summer sampling.

Ensemble Modified Aptamer Based Pattern Recognition for Adaptive Target Identification.

The difficulty of the molecular design and chemical synthesis of artificial sensing receptors restricts their diagnostic and proteomic applications. Herein, we report a concept of "ensemble modified aptamers" (EMAmers) that exploits the collective recognition abilities of a small set of protein-like side-chain-modified nucleic acid ligands for discriminative identification of molecular or cellular targets. Different types and numbers of hydrophobic functional groups were incorporated at designated positions on nucleic acid scaffolds to mimic amino acid side chains. We successfully assayed 18 EMAmer probes with differential binding affinities to seven proteins. We constructed an EMAmer-based chemical nose sensor and demonstrated its application in blinded unknown protein identification, giving a 92.9% accuracy. Additionally, the sensor is generalizable to the detection of blinded unknown bacterial and cellular samples, which enabled identification accuracies of 96.3% and 94.8%, respectively. This sensing platform offers a discriminative means for adaptive target identification and holds great potential for diverse applications.

134. Characterization of circulating clinical multi-drug resistant and methicillin resistant Staphylococcus aureus isolates in Peru

Abstract Background Multi-drug resistant and methicillin resistant Staphylococcus aureus (MDR-SA and MRSA, respectively) constitute a significant threat to hospitalized patients. Surveillance for antibiotic resistance is needed in settings where over-the-counter antibiotic use is widespread to identify increasing antibiotic resistance that limits treatment options of nosocomial infections. Methods We performed phenotypic and genotypic characterizations of MDR-SA prospectively obtained from hospitals in Lima, Peru between 2015 - 2018. We collected 3103 bacterial isolates from clinical specimens. Bacterial samples were identified by conventional microbiology and BD Phoenix, and antimicrobial susceptibility testing (AST) was performed by disc diffusion, E-test and BD Phoenix. Additionally, multiplex PCR detected mecA, femA, and lukS in MDR-SA isolates. We selected 80 S. aureus isolates that were resistant to ≥ 3 antimicrobial classes and/or resistant to oxacillin or cefoxitin for genomic characterization using an Illumina NextSeq 500 sequencer. Results Testing identified 232/3103 isolates as S. aureus, and 80/232 (34%) isolates were MDR-SA, resistant to up to nine different antimicrobial classes. We observed high proportions of resistance to aminopenicillins 80/80 (100%), macrolides 71/80 (89%), lincomycins 69/80 (86%), quinolones 66/80 (83%), aminoglycosides 38/46 (82%). Notably, 75/80 (94%) isolates were MRSA. Interestingly, AST revealed that 81% had inducible resistance to clindamycin. In contrast, all isolates were susceptible to vancomycin, linezolid, and daptomycin. Multiplex PCR revealed that 5% of isolates carried the lukS gene and the mecA gene. 76 % of the isolates carried a bifunctional aminoglycoside modifying enzyme and a aminoglycoside O-nucleotidyltransferase enzymes that confer resistance to aminoglycosides. We detected the ermA gene in 79% of the isolates. In silico multilocus sequence typing (MLST) assigned the sequence type 5 to 91.2% MRSA isolates with multiple high related clusters observed, indicating the possibility of nosocomial dissemination and possible outbreak events. Conclusion Antibiotic pressure on the resistance patterns of S. aureus is evident in the high levels of resistance seen in these clinical isolates. Disclosures All Authors: No reported disclosures.

Magnetorheological elastomer and smartphone enable microfluidic biosensing of foodborne pathogen

Rapid detection of foodborne pathogens is crucial to prevent the outbreaks of foodborne diseases. In this work, we proposed a novel microfluidic biosensor based on magnetorheological elastomer (MRE) and smartphone. First, micropump and microvalves were constructed by deforming the MRE under magnetic actuation and integrated into the microfluidic biosensor for fluidic control. Then, the micropump was used to deliver immune porous gold@platinum nanocatalysts (Au@PtNCs), bacterial sample, and immunomagnetic nanoparticles (MNPs) into a micromixer, where they were mixed, incubated and magnetically separated to obtain the Au@PtNC-bacteria-MNP complexes. After 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide were injected and catalyzed by the Au@PtNCs, smartphone was used to measure the color of the catalysate for quantitative analysis of target bacteria. Under optimal conditions, this biosensor could detect Salmonella typhimurium quantitatively and automatically in 1 h with a linear detection range of 8.0 × 101 CFU/mL to 8.0 × 104 CFU/mL and a detection limit of 62 CFU/mL. The microfluidic biosensor was compact in size, simple to use, and efficient for detection, and might be used for in-field screening of foodborne pathogens to prevent food poisoning.

SYNTHESIS, CHARACTERIZATION, AND ACTIVITY ASSESSMENT OF AMOXICYLIN/CuO COMPOUNDS AGAINST PATHOGENIC BACTERIA

Infection is a disease caused by bacteria. Some bacteria that can cause infection are Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Bacillus subtilis. Infection can be prevented by antibiotic therapy. Excessive use of antibiotics such as misuse of indications, free use in the community, inappropriate doses, and timing of application will cause new problems such as increasing bacterial resistance to antibiotics. The prevalence of drug-resistant bacteria is increasing in many parts of the world. However, this increase is accompanied by a downward trend in the development of new antibiotics. Thus, to overcome this problem new antibiotics have to be developed. This study aimed to synthesize a new antibiotic, namely to test amoxicillin/CuO and its antibacterial activity against E. coli, S. typhi, S. aureus, and B. subtilis. Antibacterial activity test using disc diffusion method. The XRD characterization results showed that the diffraction peaks of amoxicillin/CuO in the image above started from 21.13; 29.54; 30.67; 36.82; 39.5; 42.05; and 47.88. The SEM results of the Amoxicillin/CuO compound showed that the particle size of the compound was still in the form of a graph of 196-345 nm. The results of testing the antibacterial activity of amoxicillin/CuO compounds against E. coli, S. typhi, B. subtilis, and S. aureus showed that amoxicillin/CuO compounds had activity against all bacterial samples. Thus, amoxicillin/CuO compounds can be used as antibacterial therapy.

A Selective Fluorescent l-Lactate Biosensor Based on an l-Lactate-Specific Transcription Regulator and Förster Resonance Energy Transfer.

Selective detection of l-lactate levels in foods, clinical, and bacterial fermentation samples has drawn intensive attention. Many fluorescent biosensors based on non-stereoselective recognition elements have been developed for lactate detection. Herein, the allosteric transcription factor STLldR from Salmonella enterica serovar Typhimurium LT2 was identified to be stereo-selectively respond to l-lactate. Then, STLldR was combined with Förster resonance energy transfer (FRET) to construct a fluorescent l-lactate biosensor FILLac. FILLac was further optimized by truncating the N- and C-terminal amino acids of STLldR between cyan and yellow fluorescent proteins. The optimized biosensor FILLac10N0C exhibited a maximum emission ratio change (ΔRmax) of 33.47 ± 1.91%, an apparent dissociation constant (Kd) of 6.33 ± 0.79 μM, and a limit of detection of 0.68 μM. FILLac10N0C was applied in 96-well microplates to detect l-lactate in bacterial fermentation samples and commercial foods such as Jiaosu and yogurt. The quantitation results of FILLac10N0C exhibited good agreement with that of a commercial l-lactate biosensor SBA-40D bioanalyzer. Thus, the biosensor FILLac10N0C compatible with high-throughput detection may be a potential choice for quantitation of l-lactate in different biological samples.

DETECTION OF SALMONELLA TYPHIMURIUM BACTERIA ON BAKERY PRODUCTS SAMPLES USING BOILING ISOLATION TECHNIQUE

Detection of Salmonella typhimurium ATCC 14028 bacteria in bakery product samples by real-time PCR using boiling isolation technique. The basis of this research is to have an impact on economic value in carrying out the confirmation test for Salmonella typhimurium ATCC 14028, where testing is carried out conventionally will require large costs, so it is necessary to innovate in terms of modifying the testing phase so that it is more effective and efficient. The purpose of this study was to see whether the boiling isolation technique could be used for the detection test for Salmonella typhimurium ATCC 14028 on bacterial product samples. The sample in this study consisted of 15 types of bacterial product samples spiked with Salmonella typhimurium ATCC 14028 cultures that had been cultured into phase 2 working cultures. The method used in this study was qPCR analysis using the SYBR Green method. The results of real-time PCR analysis obtained Ct values in the range 7.55 - 8.91 with an average of 8.28 and a Tm value in the range 85.50 - 86.20 with an average of 85.77 Based on these data it can be concluded that the detection of Salmonella typhimurium bacteria ATCC 14028 with real-time PCR using boiling isolation technique can be applied for testing on bakery product samples.

Bacterial Biodegradation of Congo Red Dye Using Local Bacterial Isolates

The current study aims to get local bacterial isolates isolated from wastewater samples, w the highest ability to decolourize one selected Azo dye (Congo red as a model), and then test its ability to decompose Congo red dye, to obtain the most efficient bacterial isolate Four samples of wastewater collected from sewage transport pipes were used for the bacterial isolation. Forty-two bacterial isolates were obtained after inoculating these samples in the liquid MS medium, pH 7 with 1% glucose, and then on solid MS medium supplemented with50 ppm of Congo red dye. Results from primary tests showed that only eighteen bacterial isolates own varying abilities to decolourize Congo red dye and the isolates WR7, WR18 and WR30 give high clear zones (20 mm). The secondary screening was achieved to determine the most efficient isolates to degrade the Congo red dye. The results indicated that the isolates WR7, WR18, and WR30 appeared to have the highest ability to decolorize Congo red dye reaching 88.6%, 83.9%, and 92.8%, respectively. The results from the optimum conditions experiment revealed that the isolates WR7, WR18 and WR30 appeared to have the highest ability to degrade dye reached 88.7%, 83.9%, and 92.9% respectively when 100 ppm of Congo red dye was used after 3 days of incubation as compared with other studied concentrations. While the second step in this experiment confirmed that the isolate WR30 has the highest percentage to degrade the Congo red dye during all incubation periods, which reached 14.6%, 42.7%, 92.9%, 92.4% and 91.8% after 1, 2, 3, 4, and 5 days of incubation when 100 ppm of Congo red dye was used respectively as compared with WR7 and WR18 isolates which showed lower values of degradation ​​for all studied periods. The three isolates which have the highest capability to decolorize Congo red dye were identified according to morphological, physiological and biochemical tests. The results indicated that the isolates WR7, WR18 and WR30 were Bacillus subtilis, Pseudomonas aeruginosa, and Bacillus cereus respectively.

Rapid, ultrasensitive, and highly specific identification of Brucella abortus utilizing multiple cross displacement amplification combined with a gold nanoparticles-based lateral flow biosensor.

Brucella abortus (B. abortus) as an important infectious agent of bovine brucellosis cannot be ignored, especially in countries/regions dominated by animal husbandry. Thus, the development of an ultrasensitive and highly specific identification technique is an ideal strategy to control the transmission of bovine brucellosis. In this report, a novel detection protocol, which utilizes multiple cross displacement amplification (MCDA) combined with a gold nanoparticles-based lateral flow biosensor (AuNPs-LFB) targeting the BruAb2_0168 gene was successfully devised and established for the identification of B. abortus (termed B. abortus-MCDA-LFB). Ten specific primers containing engineered C1-FAM (carboxyfluorescein) and D1-biotin primers were designed according to the MCDA reaction mechanism. These genomic DNA extracted from various bacterial strains and whole blood samples were used to optimize and evaluate the B. abortus-MCDA-LFB assay. As a result, the optimal reaction conditions for the B. abortus-MCDA-LFB assay were 66°C for 40 min. The limit of detection of the B. abortus-MCDA-LFB was 10 fg/μl (~3 copies/μl) for genomic DNA extracted from pure cultures of B. abortus isolate. Meanwhile, the B. abortus-MCDA-LFB assay accurately identified all tested B. abortus strains, and there was no cross-reaction with non-B. abortus pathogens. Moreover, the detection workflow of the B. abortus-MCDA-LFB assay for whole blood samples can be completed within 70 min, and the cost of a single test is approximately 5.0 USD. Taken together, the B. abortus-MCDA-LFB assay is a visual, fast, ultrasensitive, low-cost, easy-to-operate, and highly specific detection method, which can be used as a rapid identification tool for B. abortus infections.

Streptococcus mutans Proteases Degrade Dentinal Collagen.

Here, we explored the role of S. mutans's whole cell and discrete fractions in the degradation of type I collagen and dentinal collagen. Type I collagen gels and human demineralized dentin slabs (DS) were incubated in media alone or with one of the following: overnight (O/N) or newly inoculated (NEW) cultures of S. mutans UA159; intracellular proteins, supernatant or bacterial membranes of O/N cultures. Media from all groups were analyzed for protease-mediated release of the collagen-specific imino acid hydroxyproline. Images of type I collagen and DS were analyzed, respectively. Type I collagen degradation was highest for the supernatant (p &lt; 0.05) fractions, followed by intracellular components and O/N cultures. Collagen degradation for DS samples was highest for O/N samples, followed by supernatant, and intracellular components (p &lt; 0.05). There was lower detectable degradation for both type I collagen and DS from NEW culture samples (p &lt; 0.05), and there was no type I collagen or DS degradation detected for bacterial membrane samples. Structural changes to type I collagen gel and dentinal collagen were observed, respectively, following incubation with S. mutans cultures (O/N and NEW), intracellular components, and supernatant. This study demonstrates that intracellular and extracellular proteolytic activities from S. mutans enable this cariogenic bacterium to degrade type I and dentinal collagen in a growth-phase dependent manner, potentially contributing to the progression of dental caries.

Evaluation of a Sequential Antibiotic Treatment Regimen of Ampicillin, Ciprofloxacin and Fosfomycin against Escherichia coli CFT073 in the Hollow Fiber Infection Model Compared with Simultaneous Combination Treatment.

Employ the hollow fiber infection model (HFIM) to study sequential antibiotic administration (ampicillin, ciprofloxacin and fosfomycin) using human pharmacokinetic profiles to measure changes in the rate of antibiotic resistance development and compare this to simultaneous combination therapy with the same antibiotic combinations. Escherichia coli CFT073, a clinical uropathogenic strain, was exposed individually to clinically relevant pharmacokinetic concentrations of ampicillin on day 1, ciprofloxacin on day 2 and fosfomycin on day 3. This sequence was continued for 10 days in the HFIM. Bacterial samples were collected at different time points to enumerate total and resistant bacterial populations. The results were compared with the simultaneous combination therapy previously studied. Sequential antibiotic treatment (ampicillin-ciprofloxacin-fosfomycin sequence) resulted in the early emergence of single and multi-antibiotic-resistant subpopulations, while the simultaneous treatment regimen significantly delayed or prevented the emergence of resistant subpopulations. Sequential administration of these antibiotic monotherapies did not significantly delay the emergence of resistant subpopulations compared to simultaneous treatment with combinations of the same antibiotics. Further studies are warranted to evaluate different sequences of the same antibiotics in delaying emergent resistance.

Optimization of Ultrasonic Extraction of Nutraceutical and Pharmaceutical Compounds from Bee Pollen with Deep Eutectic Solvents Using Response Surface Methodology.

In recent years, there has been increasing interest in green extraction methods and green solvents due to their many advantages. In this study, the effects of an ultrasonic extraction method and deep eutectic solvents (DESs) on the extraction of different bioactive substances from bee pollen were investigated. In this regard, the effects of process variables such as the molar ratio of the DES (1, 1.5, and 2), sonication time (15, 30, and 45 min), and ultrasonic power (90, 135 and 180 W) on total individual amino acids, total individual organic acids, and total individual phenolic compounds were investigated by response surface methodology (RSM). The optimal conditions were found to be a molar ratio of 2, sonication time of 45 min, and ultrasonic power of 180 W (R2 = 0.84). Extracts obtained via the maceration method using ethanol as a solvent were evaluated as the control group. Compared with the control group, the total individual amino acid and total individual organic acid values were higher using DESs. In addition, compounds such as myricetin, kaempferol, and quercetin were extracted at higher concentrations using DESs compared to controls. The results obtained in antimicrobial activity tests showed that the DES groups had broad-spectrum antibacterial effects against all bacterial samples, without exception. However, in yeast-like fungus samples, this inhibition effect was negligibly low. This study is the first to evaluate the impact of DESs on the extraction of bioactive substances from bee pollen. The obtained results show that this innovative and green extraction technique/solvent (ultrasonic extraction/DES) can be used successfully to obtain important bioactive compounds from bee pollen.

Bacterial plasmid-associated and chromosomal proteins have fundamentally different properties in protein interaction networks

Plasmids facilitate horizontal gene transfer, which enables the diversification of pathogens into new anatomical and environmental niches, implying that plasmid-encoded genes can cooperate well with chromosomal genes. We hypothesise that such mobile genes are functionally different to chromosomal ones due to this ability to encode proteins performing non-essential functions like antimicrobial resistance and traverse distinct host cells. The effect of plasmid-driven gene gain on protein–protein interaction network topology is an important question in this area. Moreover, the extent to which these chromosomally- and plasmid-encoded proteins interact with proteins from their own groups compared to the levels with the other group remains unclear. Here, we examined the incidence and protein–protein interactions of all known plasmid-encoded proteins across representative specimens from most bacteria using all available plasmids. We found that plasmid-encoded genes constitute ~ 0.65% of the total number of genes per bacterial sample, and that plasmid genes are preferentially associated with different species but had limited taxonomical power beyond this. Surprisingly, plasmid-encoded proteins had both more protein–protein interactions compared to chromosomal proteins, countering the hypothesis that genes with higher mobility rates should have fewer protein-level interactions. Nonetheless, topological analysis and investigation of the protein–protein interaction networks' connectivity and change in the number of independent components demonstrated that the plasmid-encoded proteins had limited overall impact in > 96% of samples. This paper assembled extensive data on plasmid-encoded proteins, their interactions and associations with diverse bacterial specimens that is available for the community to investigate in more detail.

Microbiome Analysis of the Bamboo Aphid Melanaphis bambusae Infected with the Aphid Obligate Pathogen Conidiobolus obscurus (Entomophthoromycotina).

Insect-associated microbes exert diverse effects on host fitness. This study provides insights into the microbiota of the bamboo aphid, Melanaphis bambusae, and their response to Conidiobolus obscurus infection. 16S rRNA and ITS sequencing data were used to analyze the bacterial and fungal samples associated with healthy, infected, and starved aphids. At ≥97% nucleotide similarity, the total reads were clustered into 79 bacteria and 97 fungi operational Taxonomic Units (OTUs). The phyla Proteobacteria and Ascomycota dominated the bacterial and fungal communities, respectively. The significant divergence in OTU distribution presented differential profiles of the microbiota in response to host conditions. Lower α-diversity indices were found in bacterial and fungal diversity when the aphids were experiencing fungal infection and starvation stresses, respectively. The β-diversity analyses of the communities showed significant differences among the three host conditions, demonstrating that aphid-associated microbiota could significantly shift in response to varying host conditions. Moreover, some OTUs increased under fungal infection, which potentially increased aphid susceptibility. Presumably, C. obscurus infection contributed to this increase by causing the disintegration of host tissues other than host starvation. In conclusion, understanding the differentiation of aphid microbiota caused by fungal entomopathogens helped facilitate the development of novel pest management strategies.

Metagenomic nanopore sequencing of ocular microbiome in patients with meibomian gland dysfunction.

To explore the composition of the ocular microbiome in patients with Meibomian gland dysfunction (MGD) using metagenomic nanopore sequencing. A total of 98 participants were recruited from September to December 2021, including 86 patients with MGD and 12 controls. Symptoms and signs of dry eye were assessed, and bacterial samples in the conjunctival sac (CS) and meibomian gland (MG) secretions were then identified by bacterial culture identification and metagenomic nanopore sequencing. The positive rate of CS bacterial culture in the MGD group was significantly higher than that in the normal group. A more complex composition of bacterial genera was detected in the mild and moderate MGD groups than in the control. However, the severe MGD groups had the simplest composition of bacteria. Metagenomic nanopore sequencing detected more species of bacteria than traditional culture. The CS and MG of MGD patients may have different degrees of bacterial microbiota imbalance. Metagenomic nanopore sequencing technology provides a new way for us to understand the composition of "real-world" ocular surface microorganisms.

A Sensitive GC-MS Method for Quantitation of Lipid A Backbone Components and Terminal Phosphate Modifications.

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria, serves as a target for cationic antimicrobial peptides, such as polymyxins. Membrane stress from polymyxins results in activation of two-component regulatory systems that produce lipid A modifying enzymes. These enzymes add neutral moieties, such as aminoarabinose (AraN) and ethanolamine (EtN) to lipid A terminal phosphates that mask the phosphate's negative charge and inhibit electrostatic interaction with the cationic polymyxins. Currently, these modifications may be detected by MALDI-TOF MS; however, this analysis is only semiquantitative. Herein we describe a GC-MS method to quantitate lipid A backbone components, glucosamine (GlcN) and inorganic phosphate (Pi), along with terminal phosphate modifications AraN and EtN. In this assay, lipid A is isolated from Gram-negative bacterial samples, hydrolyzed into its individual moieties, and derivatized via methoximation followed by silylation prior to analysis via GC-MS. Changes in AraN and EtN quantity were characterized using a variety of regulatory mutants of Salmonella, revealing differences that were not detected using MALDI-TOF MS analysis. Additionally, an increase in the abundance of AraN and EtN modifications were observed when resistant Enterobacter and Escherichia coli strains were grown in the presence of colistin (polymyxin E). Lastly, increased levels of Pi were found in bisphosphorylated lipid A compared to monophosphorylated lipid A samples. Because lipid A modifications serve as indicators of polymyxin resistance in Gram-negative bacteria, this method provides the capacity to monitor polymyxin resistance by quantification of lipid A modification using GC-MS.

Fruit Enzyme Activity of Molasses Combination Against Control of S. Aureus and E. Coli Bacteria

This study aims to determine the effect of the pineapple and orange peel enzymes combined with molasses on the effectiveness of the inhibition zones of gram positive (S. Aureus) and gram negative (E. Coli) bacteria. The study was conducted using the disc method, where the bacterial samples used were at a concentration of 200 millimicrons. And the addition of enzyme solution with a concentration of 20 millimicrons. In observations using Scan 500, the results obtained in samples of pineapple peel with the highest inhibition zones for E. Coliand S. Aureusbacteria, respectively, of 11.7 mm and 12.8 mm. Meanwhile, in the test of orangepeel samples, the highest inhibition zones for E. Coliand S. Aureuswere 16.6 mm and 13.2 mm, respectively. In addition, the highest pH testing of each sample of orange peel and pineapple was obtained on the third fermentation with values of 4.63 and 4.39.