Evaluation of Gradient Diffusion in vitro Susceptibility Testing of Aerococcus urinae.

OBJECTIVES: Aerococcus urinae antimicrobial susceptibility testing (AST) can be performed via broth microdilution (BMD) with Mueller-Hinton broth supplemented with lysed horse blood (MHB-LHB). We sought to compare this to the commonly used gradient diffusion method. METHODS: We compared BMD with MHB-LHB to gradient diffusion via MH agar supplemented with sheep blood for 190 A. urinae isolates against sixteen antimicrobials. RESULTS: No antimicrobials demonstrated over 90% essential and categorical agreement (EA and CA) and less than 3% major and very major error rates (ME and VME). Trimethoprim-sulfamethoxazole (TMP-STX) demonstrated an 81% major error rate and ceftriaxone demonstrated a 76% very major error rate. Agar dilution (AD) with lysed horse blood was performed for TMP-STX against 94 isolates and found 100% susceptibility, consistent with previous studies. CONCLUSIONS: Our findings cannot support the routine use of gradient diffusion with MH agar supplemented with sheep blood for A. urinae in lieu of the CLSI method given its limitations in detecting resistant strains. Our results suggest A. urinae is usually susceptible to penicillin, linezolid, tetracycline, and vancomycin. Future studies should evaluate alternative testing methods for clinical microbiology laboratories.

Calculated Drug Concentrations in Currently Available Intravitreal Therapies: Determination of Dilution Factor and Deviation From Recommended Doses.

In ophthalmology, intravitreal therapies are currently not personalized/customized and are not adjusted to the individual vitreous volume. With reference to the recently published calculation formula for a more accurate estimation of the vitreous body, we determined the dose of intravitreal medication for different vitreous volumes and compared them with the average volume. Using the axial length of the eye, the formula for the vitreous volume exact (VIVEX) can provide a more accurate indication of the vitreous volume in individual cases than an assumed standard volume of 4 mL. The concentration of active substances in small eyes may be twice as high as that in normal-sized emmetropic eyes. In contrast, large eyes may show less than half of the recommended drug concentration. The calculated concentrations of the investigated intravitreal drugs in small and large eyeballs showed impressive differences with large deviations from the recommended doses. Further systematic studies should follow to find out whether this has any impact on the effectiveness or side effects of the injected drugs.

Characterization of the key aroma compounds in different varieties of hops by application of the Sensomics approach.

Aroma is a crucial indicator of hop quality. This study analyzed the differences in aroma compound composition among six hop varieties from three regions: North America, Europe, and Asia. Descriptive analysis and sensomic approaches including gas chromatography-olfactometry/aroma extract dilution analysis, odour activity value calculation and aroma recombination were used for the detailed characterization and comparative analysis of hop aroma. A total of 55 aroma-active compounds were identified. Among them, linalool, geraniol, ß-myrcene, 2-undecanone, and methyl decanoate contributed significantly to hop aroma. Orthogonal partial least squares discriminant analysis revealed that, except for the SAAZ and XinYuan hops with some similarities in their aroma composition, the remaining hops exhibited unique aroma characteristics. A total of 16 compounds, including methyl 5-methylhexanoate and (E)-ß-farnesene, were identified as differentiating aroma compounds in the six hop samples. This study enriches the knowledge on hop flavour with different origins and provides valuable insights into its application.

A comparative study to determine the key aroma components of yogurt aroma types based on Sensomics and Flavoromics.

This study used Sensomics to examine four previously obtained yogurt aroma type profiles. 14 key aroma-active compounds were identified as significant contributors (p ≤ 0.05) in the four aroma types using gas chromatography-mass spectrometry-olfactometry (GC-MS/O), aroma extract dilution analysis (AEDA), odor activity values (OAV), and aroma recombination and omission experiments. The Sensomics and previous Flavoromics results were compared, showing that Flavoromics identified 10 indicator compounds for distinguishing aroma types. Eight were the same as the key aroma-active compounds identified via Sensomics, namely acetic acid, pentanoic acid, decanoic acid, 3-hydroxy-2-butanone, 2,3-pentanedione, acetaldehyde, δ-decalactone, and dimethyl sulfone. Sensomics revealed a prominent similarity between the categories of key aroma-active compounds of the four aroma types, with a higher sensory contribution. Flavoromics showed less overlapping between the indicator compounds, mainly related to the distinction between the four aroma types. Sensomics and Flavoromics serve distinct research objectives and should be selected according to the study subject.

Hyposalinity elicits physiological responses and alters intestinal microbiota in Korean rockfish Sebastes schlegelii.

Global warming significantly impacts aquatic ecosystems, with changes in the salt environment negatively affecting the physiological responses of fish. We investigated the impact of hyposalinity on the physiological responses and intestinal microbiota of Sebastes schlegelii under the context of increased freshwater influx due to climate change. We focused on the osmoregulatory capacity, oxidative stress responses, and alterations in the intestinal microbiome of S. schlegelii under low-salinity conditions. Our findings revealed compromised osmoregulatory capacity in S. schlegelii under low-salinity conditions, accompanied by the activation of oxidative stress responses, indicating physiological adaptations to cope with environmental stress. Specifically, changes in Na+/K+-ATPase (NKA) activity in gill tissues were associated with decreased osmoregulatory capacity. Furthermore, the analysis of the intestinal microbiome led to significant changes in microbial diversity. Exposure to low-salinity environments led to dysbiosis, with notable decreases in the relative abundance of Gammaproteobacteria at the class level and specific genera such as Enterovibrio, and Photobacterium. Conversely, Bacilli classes, along with genera like Mycoplasma, exhibited increased proportions in fish exposed to low-salinity conditions. These findings underscore the potential impact of environmental salinity changes on the adaptive capacity of fish species, particularly in the context of aquaculture. Moreover, they highlight the importance of considering both physiological and microbial responses in understanding the resilience of aquatic organisms to environmental stress. Additionally, they highlight the importance of intestinal microbiota analyses in understanding the immune system and disease management in fish.

Effect of mineral and organic fertilizer on N dynamics upon erosion-induced topsoil dilution.

Erosion-induced topsoil dilution strongly affects cropland biogeochemistry and is associated with a negative effect on soil health and crop productivity. While its impact on soil C cycling has been widely recognized, there is little information about its impact on soil N cycling and N fertilizer dynamics. Here, we studied three factors potentially influencing N cycling and N fertilizer dynamics in cropping systems, namely: 1.) soil type, 2.) erosion-induced topsoil dilution and 3.) N fertilizer form, in a full-factorial pot experiment using canola plants. We studied three erosion affected soil types (Luvisol, eroded Luvisol, calcaric Regosol) and performed topsoil dilution in all three soils by admixing 20 % of the respective subsoil into its topsoil. N fertilizer dynamics were investigated using either mineral (calcium ammonium nitrate) or organic (biogas digestate) fertilizer, labeled with 15N. The fertilizer 15N recovery and the distribution of the fertilizer N in different soil fractions was quantified after plant maturity. Fertilizer N dynamics and utilization were influenced by all three factors investigated. 15N recovery in the plant-soil system was higher and fertilizer N utilization was lower in the treatments with diluted topsoil than in the non-diluted controls. Similarly, plants of the organic fertilizer N treatments took up significantly less fertilizer N in comparison to mineral fertilizer treatments. Both topsoil dilution and organic fertilizer application promoted 15N recovery and N accumulation in the soil fractions, with strong differences between soil types. Our study reveals an innovative insight: topsoil dilution due to soil erosion has a negligible impact on N cycling and dynamics in the plant-soil system. The crucial factors influencing these processes are found to be the choice of fertilizer form and the specific soil type. Recognizing these aspects is essential for a precise and comprehensive assessment of the environmental continuum, emphasizing the novelty of our findings.

Potential effects of host competence and schooling behavior on parasite transmission in a host-pathogen system: a test of the dilution effect.

High species diversity in a community may reduce the risk of infectious disease, termed the dilution effect. However, the generality of the dilution effect in different disease systems remains controversial as both host competence and behaviors of hosts may play roles in dilution or amplification of disease. Using the goldfish (Carassius auratus)-monogenean ectoparasite (Gyrodactylus kobayashii) system, effects of host competence and schooling behavior on parasite transmission were investigated while holding focal host density constant. Following competency tests of 12 fish species as potential hosts for the parasite, infection by G. kobayashii was determined on fins of goldfish mixed with each of three different species based on their level of host competence, including Prussian carp, Carassius gibelio (low competence), grass carp, Ctenopharyngodon idellus (non-competent), swordtail, Xiphophorus helleri (non-competent), and the four species combined. Compared with mean abundance (85.8 ± 25.1) on goldfish in the control group, the mean abundance on goldfish decreased significantly when paired with 10 Prussian carp (30.0 ± 16.5), but did not differ significantly when paired with 10 swordtail (70.0 ± 22.2), 10 grass carp (116.1 ± 33.2), or the multi-species of three Prussian carp, four grass carp and three swordtail (75.9 ± 30.8) during the 11-day experiment. The parasite was also found on the Prussian carp in the Prussian carp group and the multi-species group at a mean abundance of 7.1 and 10.9, respectively. Video recording showed that the school of goldfish mixed well with the Prussian carp, while they maintained separation from the grass carp and swordtail when mixed together. The distance between goldfish increased, and swimming speed and contact time decreased with the additional of other fish species for all groups. The results suggested that the presence of a low-competence host in sufficient numbers was a necessary condition for a dilution effect due to encounter reduction, and the dilution effect may also be enhanced by changes in schooling behavior of goldfish in the presence of low competence hosts. However, the presence of non-competent hosts did not result in any dilution effect owing to the specialist nature of the parasites and the lack of mixing with schools of goldfish.

Identification of odor-active compounds in Nile tilapia (Oreochromis niloticus) from recirculated aquaculture systems: A case study with different depuration procedures.

Off-flavors are a major challenge for companies using recirculated aquaculture systems (RAS). In the presented work, we comprehensively characterize the odorant composition of Nile tilapia (Oreochromis niloticus) raised in RAS and compare the impact of two depuration processes on the odorant composition and aroma profile of the fish. Fish collected from the production tank and after two different tank pre-disinfection approaches in the depuration process (high pH versus H2O2) were investigated. A combined sensory-instrumental investigation revealed the presence of 115 odorants, of which 83 were successfully identified. The compounds decanal, tridecanal, (Z)-1,5-octadien-3-one, octane-2,3-dione, benzophenone, non-3-yn-1-ol, γ-dodecalactone, (Z)-geranylacetone, 2,3-diethyl-5-methylpyrazine, 1-methylpyrrolidin-2-one, 2-acetyl-2-thiazoline, benzothiazole, skatole, and 5α-androst-16-en-3-one were detected with the highest flavor dilution factors and are described for the first time as odor-active compounds in fish from RAS. The results indicate that depuration decreased the levels of 78 different odorants from the fish, including the potent earthy smelling odorants geosmin, isoborneol and 2,3-diethyl-5-methylpyrazine.

A case of atypical IgA paraprotein interference on multiple chemistry assays: How to deal with it.

This case report discusses how paraproteins interfere with multiple chemistry analyses and protocols to overcome such obstacles. A serum specimen containing two monoclonal IgA (llight chain) paraproteins is subjected to a battery of tests on three wet chemistry platforms - AU5800, Cobas Pure, and Alinityci; the results were compared with those on a Vitros 350/ ECiQ dry chemistry platform. Paraprotein interference was found to affect the bilirubins, inorganic phosphate, and iron, whose repeat runs were also found to be irreproducible. Dilution with normal saline also failed to produce a satisfactory effect. Deproteinization by polyethylene glycol and dilution of the specimen with a normal serum specimen were observed to produce desirable results. Interference by IgA paraprotein on measurement of the bilirubin, phosphate, and iron in the wet chemistry system can be mitigated either by deproteinization or by dilution with normal serum.

Assessing a Fermented Whey Beverage Biofortified with Folate as a Potential Folate Source for Humans.

Folate, a vital water-soluble vitamin (B9), requires specific attention as its recommended daily intake frequently is not reached in countries without mandatory fortification. In this regard, biofortification with microorganisms like Bifidobacterium and Streptococcus offers a compelling approach for enhancing food with natural folates. A randomized, nonblinded, and monocentric human pilot study is conducted to assess the bioavailability of a folate-biofortified fermented whey beverage, comprising 3 intervention days and a controlled replenishment phase before and during the assay. Folate plasma concentration (5-CH3-H4folate) is determined using a stable isotope dilution assay and LC-MS/MS detection. Biokinetic parameters (cmax and tmax) are determined, and areas under the curve (AUC) normalized to the basal folate plasma concentration are calculated. An average bioavailability of 17.1% in relation to the 5-CH3-H4folate supplement, ranging from 0% to 39.8%, is obtained. These results reiterate the significance of additional research into folate bioavailability in general and dairy products. Further investigations are warranted into folate-binding proteins (FBP) and other potential limiting factors within the food and individual factors. In summary, biofortification via fermentation emerges as a promising avenue for enhancing the natural folate content in dairy and other food products.

Construction of a Calibration Curve for Lycopene on a Liquid-Handling Platform─Wider Lessons for the Development of Automated Dilution Protocols.

Liquid-handling is a fundamental operation in synthetic biology─all protocols involve one or more liquid-handling operations. It is, therefore, crucial that this step be carefully automated in order to unlock the benefits of automation (e.g., higher throughput, higher replicability). In the paper, we present a study, conducted at the London Biofoundry at SynbiCITE, that approaches liquid-handling and its reliable automation from the standpoint of the construction of the calibration curve for lycopene in dimethyl sulfoxide (DMSO). The study has important practical industrial applications (e.g., lycopene is a carotenoid of industrial interest, DMSO is a popular extractant). The study was also an effective testbed for the automation of liquid-handling. It necessitated the development of flexible liquid-handling methods, which can be generalizable to other automated applications. In addition, because lycopene/DMSO is a difficult mix, it was capable of revealing issues with automated liquid-handling protocols and stress-testing them. An important component of the study is the constraint that, due to the omnipresence of liquid-handling steps, errors should be controlled to a high standard. It is important to avoid such errors propagating to other parts of the protocol. To achieve this, a practical framework based on regression was developed and utilized throughout the study to identify, assess, and monitor transfer errors. The paper concludes with recommendations regarding automation of liquid-handling, which are applicable to a large set of applications (not just to complex liquids such as lycopene in DMSO or calibration curves).

Evaluation of the Accuracy of Estimated Endpoint Titer of NOVA View in Indirect Immunofluorescent Antinuclear Antibody Testing.

For antinuclear antibody (ANA) screening, the gold standard method is an indirect immunofluorescence assay (IIFA) using HEp-2 cells, and a serial dilution test is needed to determine the endpoint titer. We aimed to evaluate the accuracy of the estimated endpoint titer (eEPT) by the NOVA View system, by comparing it with the EPT by the serial dilution method (dEPT). The endpoint titers of a total of 1518 ANA positive cases with five major patterns including speckled, homogeneous, centromere, nucleolar, and nuclear dots patterns were determined using both the estimation function and the serial dilution method by the NOVA View system. A significant correlation between the light intensity unit (LIU) values and dEPTs was identified in all five patterns with high ρ values, ranging from 0.666 to 0.832. However, the overall exact match rate between dEPT and eEPT was 22.1% (336/1518), with the ±one-titer match rate being highest in the centromere pattern (62.8%, 81/129), and lowest in the homogeneous pattern (37.6%, 200/532). This suggests that while LIU values correlate well with dEPT, there are discrepancies in numerical agreement. Most cases that did not show an exact match, showed one-to-three-titer overestimations by eEPT. Therefore, adjusting eEPT downward significantly improved the concordance rates with dEPTs. Further investigation for an appropriate cutoff of LIU values for determining eEPT should be performed for clinical application and contribution to the standardization of the ANA titer.

Neisseria gonorrhoeae isolates resistant to extended spectrum cephalosporins and macrolides isolated from symptomatic men in western Kenya.

BACKGROUND: We characterized the antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae (NG) isolated from symptomatic men at a sexually transmitted infection clinic in Kisumu, Kenya. METHODS: Two urethral swabs were obtained from symptomatic men between 2020 and 2022, one for Gram's stain and the other inoculated directly onto modified Thayer-Martin media containing 1% VCNT and 1% IsoVitaleX enrichment. Culture results were confirmed by colony morphology, Gram's stain and oxidase test. Duplicate isolates were shipped to Uniformed Services University for confirmation and characterization. Susceptibility to eight drugs was assessed by E-test. Agar dilution confirmed resistance to ceftriaxone, cefixime, and azithromycin. Susceptibility, intermediate resistance (IR), and resistance (R) were determined according to published criteria. RESULTS: Of 154 enrolled participants, 112 were culture-positive for NG. Agar dilution results in 110 (98.2%) showed the following: azithromycin-R (1.8%), and 4.5% R or IR to ceftriaxone or cefixime: ceftriaxone-R (0.9%), ceftriaxone-IR (2.7%), and cefixime-IR (2.7%). By E-test, most isolates were IR or R to tetracycline (97.2%), penicillin (90.9%), and ciprofloxacin (95.4%). CONCLUSIONS: We detected NG with resistance to azithromycin and ceftriaxone, indicating a growing threat to the current Kenyan dual syndromic treatment of urethritis with cephalosporin plus macrolides. Ongoing AMR surveillance is essential for effective drug choices.

A semi-mechanistic aging model of Pb added to soil by a modified stable isotope dilution technique.

The aging of Pb added to soils has not been studied by the isotopic technology because of difficulties in determination of isotopically exchangeable Pb in soil, so that a set of 10 typical agricultural soils in China and a one-year aging experiment with the addition of water-soluble Pb to the soils were carried out. A modified stable isotope dilution technique to determine isotopically exchangeable Pb in soil was developed where 0.2 mM EDTA (ethylenediaminetetraacetic acid) as the extractant. When water-soluble Pb was added to soil, the isotopically exchangeable Pb (Eadd%, the percentage of isotopically exchangeable Pb to total Pb added to soil) initially decreased rapidly and gradually slowly. A semi-mechanistic aging model of Pb added to soils, including precipitation/nucleation (Y1), micropore diffusion (Y2), and organic matter encapsulation processes (Y3) was developed with the root mean square error 8.3% where Y1, Y2, and Y3 accounted for 0.02~26.9%, 1.4~21.8% and 3.8~11.3%, respectively, when the pH 4.0~8.0 and organic matter 2.0~6.0%. Soil pH was a vital factor affecting the aging rate. When the pH increased by 1 unit, the Eadd value decreased by approximately 9%. The model could be used to scale ecotoxicological data of Pb in soil generated in different aging times.

Nasal exudate for diagnosis of stroke: fundamental studies through iron fractionation, total iron, and targeted protein determinations.

During the last years, there has been an increasing research interest in the analysis of biological fluids requiring non-invasive sampling for biomedical and clinical applications. In this work, we have focused on the nasal exudate with the aim of investigating the potential use of this fluid to know the role of iron in stroke and also for diagnosis. Potential differences in the nasal exudate, collected in swabs, from diagnosed hemorrhagic stroke, ischemic stroke, and control groups were investigated with regard to total iron by inductively coupled plasma-mass spectrometry, iron fractionation studies by size exclusion chromatography together with post-column isotope dilution analysis, and four proteins containing iron (ferritin, transferrin, lactoferrin, and ferroportin) with ELISA kits. All these analyses represent an analytical challenge, considering the rather limited amount of sample (10-40 mg) available, being the nasal exudate extracted from the swab with 300 µL 10 mM Tris/HCl, pH = 7.4. Studies to obtain reliable analytical information, such as the blank contribution of the sampling step, evaluation of the extraction efficiency of the nasal exudate from the swab, and normalization strategies for data treatment, have been carried out. Results showed that despite the limited number of investigated samples, fractionation studies as well as the concentrations of ferritin and ferroportin obtained with ELISA kits showed a differential behavior between the different cohorts.

Combination of Standard Addition and Isotope Dilution Mass Spectrometry for the Accurate Determination of Melamine and Cyanuric Acid in Infant Formula.

In the melamine scandals of the early 2000s, different companies of the dairy industry cheated their products by applying chemical substances to feign a higher content of nitrogen. However, this had a severe toxic impact on the kidney health of consumers. As a result, tremendous effort was put into the prevention of further harm to the public. In the present study, a fast-screening method for the determination of melamine and cyanuric acid in infant formula was developed. While a 1D-LC approach is faster and easier to set up, a 2D-LC approach allows for a more accurate result with better selectivity and sensitivity. For both instrumental approaches, the signal ratio of the isotopologues was crucial and had a dominant effect on the results and the measurement uncertainty. For this reason, the different contributions to the measurement uncertainty were determined experimentally using Matched Standard Addition-IDMS and compared to the Exact Matching Double IDMS.

Diet and habitat as determinants of intestine length in fishes.

Fish biologists have long assumed a link between intestinal length and diet, and relative gut length or Zihler's index are often used to classify species into trophic groups. This has been done for specific fish taxa or specific ecosystems, but not for a global fish dataset. Here, we assess these relationships across a dataset of 468 fish species (254 marine, 191 freshwater, and 23 that occupy both habitats) in relation to body mass and fish length. Herbivores had significantly relatively stouter bodies and longer intestines than omni- and faunivores. Among faunivores, corallivores had longer intestines than invertivores, with piscivores having the shortest. There were no detectable differences between herbivore groups, possibly due to insufficient understanding of herbivorous fish diets. We propose that reasons for long intestines in fish include (i) difficult-to-digest items that require a symbiotic microbiome, and (ii) the dilution of easily digestible compounds with indigestible material (e.g., sand, wood, exoskeleton). Intestinal indices differed significantly between dietary groups, but there was substantial group overlap. Counter-intuitively, in the largest dataset, marine species had significantly shorter intestines than freshwater fish. These results put fish together with mammals as vertebrate taxa with clear convergence in intestine length in association with trophic level, in contrast to reptiles and birds, even if the peculiar feeding ecology of herbivorous fish is probably more varied than that of mammalian herbivores. Supplementary Information: The online version contains supplementary material available at 10.1007/s11160-024-09853-3.

Two novel Pleosporales species isolated from the bark of Acer saccharum.

During a survey of culturable microfungi from the bark of sugar maple (Acer saccharum), Atrocalyx glutinosus and Nigrograna rubescens, two novel species of Pleosporales (Dothideomycetes) were isolated from several locations in eastern Ontario, Canada. Formal species descriptions are presented based on unique colony phenotypes and micromorphological characteristics and supported using multi-locus molecular phylogenetic comparisons with similar species. Both A. glutinosus and N. rubescens produce pycnidial asexual morphs in culture. As their names imply, under specific culture conditions, A. glutinosus excretes large amounts of the glutinous polysaccharide pullulan and N. rubescens produces a dark red naphthoquinone pigment that diffuses in the culture medium. Citation: Mack JN, Sproule A, Shields SW, Seifert KA, Smith M, Overy DP (2024). Two novel Pleosporales species isolated from the bark of Acer saccharum . Fungal Systematics and Evolution 13: 1-14. doi: 10.3114/fuse.2024.13.01.

Isotope dilution with isotopically labeled biomass: An effective alternative for quantitative metabolomics.

BACKGROUND: State-of-the-art quantitative metabolomics relies on isotope dilution using internal standards (IS) derived from fully 13C labeled biomass. By spiking samples and external standards with known amounts of IS, the spike characterization demands are kept to a minimum. In fact, it is sufficient to experimentally assess the isotopic enrichment of the IS. This study develops the yeast derived IS toolbox further, (1) by characterizing the concentration levels of hydrophilic metabolites in a yeast fermentation batch and (2) by exploring the analytical figures of merit of one-point IS versus multipoint external calibration using IS, the established gold-standard for quantitative metabolomics. RESULTS: Independent reverse isotope dilution experiments using different chromatographic methods over a period of several months, delivered a list of 83 13C-labeled metabolites with fully characterized concentration and their uncertainty, covering 5 orders of magnitude, from the nanomolar to the low millimolar range. The 13C-labeled yeast-derived IS showed excellent intermediate stability with 92 % of molecules showing inter-method RSDs ≤30 % (75 % of molecules showed RSDs ≤15 %) over a timeframe of five months. One-point internal standardization with the characterized labeled biomass achieved figures of merit equivalent to multipoint calibrations for the majority of metabolites. SIGNIFICANCE: The proposed calibration workflow rationalizes time and standard expenditure and is particularly beneficial for laboratories dealing with wide-target assays and small analysis batches. The present assessment serves as a seminal study for further developments of the concept towards absolute quantification from archive high-resolution MS data of U13C-biomass-spiked samples and the implementation of quick biomass recalibration with each experiment, promising seamless transition between internal standards derived from different fermentation batches.