Association between adenosine triphosphate luminometry of feeding equipment and environmental and health parameters of preweaning calves on dairy farms.

The objective of this study was to examine the influence of different environmental factors on ATP luminometry measurements of feeding equipment and to investigate associations with health of preweaning calves and the levels of ATP identified through luminometry. On 50 commercial dairy farms in Quebec, Canada, ATP luminometry measurements (in relative light units, RLU) were obtained using the direct swabbing technique with Hygiena UltraSnap swabs and a liquid rinsing technique with the same swab for automatic milk feeders (AMF), bottles, buckets, esophageal tube feeders (ET), milk replacer, nipples, and water. During this visit, environmental factors (including temperature, air draft, humidity, ammonia, and bacterial count) were collected, and a clinical examination (including respiratory score and fecal score) was performed for all preweaning calves present at the farm. This process was repeated 4 times in a year, leading to collection of luminometer results, environmental parameters, and overall health of calves for each season per farm. Overall, a difference in luminometer results was seen between the different periods sampled for all feeding equipment (except the ET), milk replacer, and water, showing higher RLU values in spring and summer and lower values in autumn and winter. When comparing RLU measurements with environmental factors, only a low to negligible correlation could be found. When feeding equipment was classified as not contaminated or contaminated based on previously described cutoff values, a good agreement within a farm for the different seasons was noticed only for nipples (Gwet's agreement AC1 = 0.64), with a poor to moderate agreement for other feeding equipment. Regarding the different models of nipples, Peach Teat nipples showed higher RLU values compared with Merrick's nipples. An association was seen between the proportion of preweaning calves suffering from diarrhea on the farm and the contamination of AMF based on ATP luminometry (logistic regression estimate = 0.52). For other feeding equipment, milk replacer, and water, no significant associations were found. This study showed that ATP luminometry measurements of feeding equipment from preweaning calves are susceptible to seasonality and type of nipple. Thus, these factors should be taken into consideration when interpreting results. Additionally, an association could be made between diarrhea in preweaning calves and the contamination of AMF based on ATP luminometry, showing the potential clinical importance of this on-farm hygiene assessment tool.

Miniaturizable Chemiluminescence System for ATP Detection in Water.

We present the design, fabrication, and testing of a low-cost, miniaturized detection system that utilizes chemiluminescence to measure the presence of adenosine triphosphate (ATP), the energy unit in biological systems, in water samples. The ATP-luciferin chemiluminescent solution was faced to a silicon photomultiplier (SiPM) for highly sensitive real-time detection. This system can detect ATP concentrations as low as 0.2 nM, with a sensitivity of 79.5 A/M. Additionally, it offers rapid response times and can measure the characteristic time required for reactant diffusion and mixing within the reaction volume, determined to be 0.3 ± 0.1 s. This corresponds to a diffusion velocity of approximately 44 ± 14 mm2/s.

Hygiene management practices and adenosine triphosphate luminometry of feeding equipment in preweaning calves on dairy farms in Quebec, Canada.

The objective of this study was to describe the cleaning practices currently used for preweaning calves on dairy farms in Quebec, Canada. In addition, contamination of feeding equipment for preweaning calves was described using ATP (expressed as relative light units, RLU), visual assessment, and bacteriological analysis. A questionnaire was administered on 50 commercial dairy farms in Quebec, Canada, regarding the self-reported cleaning protocol used for feeding equipment of preweaning calves. During the visit, a visual score was given to the feeding equipment available at the farm. Afterward, ATP luminometry measurements were obtained using Hygiene UltraSnap and MicroSnap swabs (Hygiene, Camarillo, CA), and the liquid rinsing technique for buckets, nipples, bottles, esophageal tube feeders (ET), the tube of automatic milk feeders (AMF), water samples, and milk replacer. An additional direct swabbing technique was performed on buckets and nipples. The fluid retrieved from the liquid rinsing technique was also used to determine the total bacterial count (TBC) and total coliform count. Based on the bacteriological analysis, optimal RLU cutoff values to determine contamination were obtained. The median (interquartile range) luminometer measurements using the UltraSnap and direct technique for buckets and nipples were 2,082 (348-7,410) and 3,462 (462-7,518) RLU, respectively; and, using the liquid technique for bottles, ET, AMF, water, and milk replacer were 43 (4-974), 15 (4-121), 301 (137-1,323), 190 (71-358), and 94 (38-218) RLU, respectively. Overall, for all equipment and both techniques used, higher RLU values were seen in UltraSnap samples compared with MicroSnap samples. Additionally, for buckets and nipples, higher RLU values were obtained for the direct swabbing method compared with the liquid sampling method for both swabs used. No differences in the level of contamination were seen between the different feeding equipment used within a farm. Overall, a higher correlation with bacteriological results was noticed for ATP luminometry compared with the visual score, with a high correlation for nipples and bottles using the UltraSnap and liquid technique. Based on the classification of "contaminated" (TBC ≥100,000 cfu/mL) or "not contaminated" (TBC <100,000 cfu/mL), optimal ATP luminometer cutoff values for buckets, nipples, bottles, AMF, water, and milk replacer were 798, 388, 469, 282, 1,432, and 93 RLU, respectively. No clear association was found between ATP measurements and the self-reported cleaning protocol. This study gave new insights into the current cleaning procedures and contamination of feeding equipment for preweaning calves on dairy farms in Quebec. In addition, ATP luminometry cutoff values could help benchmark farms regarding cleaning practices and provide customized advice, improving the overall hygiene management, and thus the health, of preweaning calves on dairy farms.

Potent Synergistic Interactions between Lopinavir and Azole Antifungal Drugs against Emerging Multidrug-Resistant Candida auris.

The limited therapeutic options and the recent emergence of multidrug-resistant Candida species present a significant challenge to human medicine and underscore the need for novel therapeutic approaches. Drug repurposing appears as a promising tool to augment the activity of current azole antifungals, especially against multidrug-resistant Candida auris In this study, we evaluated the fluconazole chemosensitization activities of 1,547 FDA-approved drugs and clinical molecules against azole-resistant C. auris This led to the discovery that lopinavir, an HIV protease inhibitor, is a potent agent capable of sensitizing C. auris to the effect of azole antifungals. At a therapeutically achievable concentration, lopinavir exhibited potent synergistic interactions with azole drugs, particularly with itraconazole against C. auris (fractional inhibitory concentration index [ΣFICI] ranged from 0.04 to 0.09). Additionally, the lopinavir/itraconazole combination enhanced the survival rate of C. auris-infected Caenorhabditis elegans by 90% and reduced the fungal burden in infected nematodes by 88.5% (P < 0.05) relative to that of the untreated control. Furthermore, lopinavir enhanced the antifungal activity of itraconazole against other medically important Candida species, including C. albicans, C. tropicalis, C. krusei, and C. parapsilosis Comparative transcriptomic profiling and mechanistic studies revealed that lopinavir was able to significantly interfere with the glucose permeation and ATP synthesis. This compromised the efflux ability of C. auris and consequently enhanced the susceptibility to azole drugs, as demonstrated by Nile red efflux assays. Altogether, these findings present lopinavir as a novel, potent, and broad-spectrum azole-chemosensitizing agent that warrants further investigation against recalcitrant Candida infections.

Development of a highly sensitive microplate luminometer using ATP bioluminescence.

Analytical techniques using ATP bioluminescence, which has a high quantum yield and substrate specificity, are widely used in various assays, such as luciferase reporter assays, in the biological sciences. Although most microplate luminometers can be used to measure ATP luminescence with 96-well or 384-well microplates, their ATP detection limits are typically several tens of amol, which is not sufficient for evaluating cell activities and variability within small samples, such as those containing only a few cells. To analyze cell activities at low ATP concentrations, a more sensitive microplate luminometer is required. Therefore, in this study, we developed an automated highly sensitive microplate luminometer that could perform reagent dispensing and bioluminescence measurement with a 96-well microplate within 10 min. ATP bioluminescence was detected by pressing a photomultiplier tube (PMT) against a microplate surface to seal the measured well with the light-receiving surface of the PMT. This enabled a high light collection efficiency and low luminescence crosstalk, defined as the intensity of stray light from an adjacent well. As a result, the ATP detection limit was 0.97 amol, and the luminescence crosstalk was 4.4 × 10-6 . Both values were one order of magnitude better than that of a typical microplate luminometer. In addition, the same gradient linearity of luminescence intensity against the ATP concentration was confirmed for both high and low ATP concentrations, and the dynamic range of our microplate luminometer was 106 . Overall, our findings demonstrated that our novel microplate luminometer may have wide application in biological sciences research.

Evaluation of the complexity of indoor air in hospital wards based on PM2.5, real-time PCR, adenosine triphosphate bioluminescence assay, microbial culture and mass spectrometry.

BACKGROUND: The aim of this study was to establish a set of assessment methods suitable for evaluating the complex indoor environment of hospital wards and to ascertain the composition of bacteria and microbial ecology of hospital wards. METHODS: Colony-forming units (CFUs), PM2.5 detection, real-time PCR, and adenosine triphosphate (ATP) bioluminescence assay were employed to evaluate the complexity of indoor air in 18 wards of nine departments in a hospital and two student dormitories in a university. Subsequently, the microbial samples were quantified and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Although the studied indices were relatively independent, the PM2.5 content was correlated with bacterial CFUs determined by passive sedimentation method, bacterial and fungal counts measured by real-time PCR, and ATP bioluminescence assay. The composition of microorganisms in the air of hospital wards differed from that in the air of student dormitories. The dominant genera in hospital wards were Staphylococcus (39.4%), Micrococcus (21.9%), Corynebacterium (11.7%), Kocuria (4.4%), Bacillus (2.9%), Streptococcus (1.6%), Moraxella (1.6%), and Enterococcus (1.3%), and the microbial ecology differed between Respiration Dept. III and other hospital departments. Additionally, 11.1 and 27.3% of bacteria in hospital wards and student dormitories were not identified, respectively. CONCLUSIONS: Assessment of environmental quality of hospital wards should be based on comprehensive analysis with multiple indicators. There may be imbalances in the microbial diversity in the hospital wards, therefore, monitoring of the environmental quality of hospitals is important in the prevention of nosocomial infections.

ATP bioluminescence assay for evaluating cleaning practices in operating theatres: applicability and limitations.

BACKGROUND: Environmental cleaning practice plays an important role in reducing microbial contamination in hospital surfaces and contributes to prevent Healthcare Associated Infections. Adenosine Triphosphate (ATP) bioluminescence assay is a commonly used method for assessing environmental cleanliness on healthcare surfaces. This study tested the feasibility of using ATP-bioluminescence assay for evaluating the efficiency of cleaning procedures in the operating theatre settings, comparing the ATP-bioluminescence test with the traditional culture method. METHODS: The surfaces of 10 operating rooms of two public hospitals (140 samples in total) were examined "at rest", in two moments of the same daily session: before the first scheduled operation (Pre), and before the second, after a clean environment was re-established (Post). Surface contamination was assessed using the cultural method to detect Total Viable Counts (TVC36°C) and ATP-bioluminescence assay (RLU). RESULTS: The examined surfaces presented very low TVCs (geometric means: 1.8 CFU/plate; IC95%: 1.6-2.0), always compliant with the relative reference standards. No statistical correlation was found between ATP values and TVCs. However, considering the results in terms of general evaluation of hygienic quality of surfaces, the two methods were consistent in identifying the most contaminated areas (Hospital A > Hospital B; Pre > Post; most contaminated surfaces: scialytic lamp). Furthermore, the ATP mean values showed a progressive increase from surfaces with TVC = 0 to surfaces with TVC > 15 CFU/plate. CONCLUSIONS: Although not an alternative to cultural methods, the ATP-bioluminescence-assay can be a useful tool to measure the efficiency of cleaning procedures also in environments with very low microbial counts. Each health facility should identify appropriate reference values, depending on the devices used and on the basis of the analysis of the data collected through spatial and temporal sampling series. By providing a rapid feedback, the ATP-assay helps to increase the awareness of operators and allows immediate action to be taken in critical situations.

Evaluation of food storage racks available on the Polish market in the hygienic context

Background: Providing safe food products to the consumer depends on the material and technology used and adherence to hygienic practices, throughout the production process. The degree of microbial contamination of a surface is an important indicator of equipment cleanliness and effectiveness of cleaning and disinfection. Used material, construction solutions and quality of the applied devices also have an effect on hygienic status. Objective: The objective of the present study was to evaluate the influence of the design and construction material of selected food storage racks, available on the Polish market, on their hygienic status. Material and methods: The study was based on determination of the capability of microbial growth on the surface of the racks and the effectiveness of their cleaning. Microbiological cleanliness on the surface of the racks was monitored by the contact plates which are able to estimate the total number of icroorganisms. Examination of effectiveness of cleaning was conducted by the use of ATP bioluminescence method. Results: This experiment has proven a significant influence of adopted construction solutions on the hygienic status of the examined racks. Presence of antibacterial layer and a choice of the appropriate construction material characterized by a low surface roughness impedes the microbial growth and increases the effectiveness of cleaning. Conclusions: Design solutions have significant impact on the hygienic status of shelves. Selection of a suitable material for the construction of racks can greatly reduce the possibility of the development of microorganism, despite the low efficiency of the cleaning. The application of antimicrobial coatings inhibits microbial growth.

Residual contamination and bioburden after reprocessing of single-use endoscopic ultrasound needles: An ex vivo study.

BACKGROUND AND AIM: Endoscopic ultrasound (EUS) aspiration needles are single-use devices. However, in many centers, because of cost-constraints, these devices are reused multiple times. We studied microbiological contamination and bioburden on reprocessed needles to evaluate whether these devices can be successfully sterilized. METHODS: We studied 10 EUS needles each of 19 G, 22 G, and 25 G in size, and five 22-G ProCore needles. After initial use, each needle was reprocessed by a standardized protocol. We used standard microbiological cultures, as well as ATP bioluminescence technique to quantify bioburden as relative light units (RLU). We defined significant soil contamination by RLU values >200. We also used extractant fluid to disrupt cell membranes in an attempt to enhance ATP detection. RESULTS: We found culture positivity in 3/34 (8.8%), and detectable bioburden on the exposed surface of 33/35 (94.3%), and inside lumen of 29 (82.9%) reprocessed FNA needles. Significant bioburden was found in three (8.6%) and two (5.7%) needles on the surface and lumen, respectively. We found that use of extractant fluid enhanced detection of bioburden. Larger (19 G) needles had higher surface contamination (P = 0.016), but there was no relation of luminal contamination with needle diameter (P = 0.138). Sheath design and presence of side bevel did not influence extent of contamination. There was significant correlation between the surface and intraluminal bioburden (P < 0.001). CONCLUSIONS: There is significant bioburden in reprocessed EUS needles; standard microbiological cultures have low sensitivity for detection of needle contamination. We have provided objective evidence for the futility of reprocessing attempts, and practice of EUS needle reuse should be discontinued.

Effectiveness of ATP bioluminescence to assess hospital cleaning: a review.

INTRODUCTION: Contamination of hospital surfaces plays an important role in the transmission of several healthcare-associated microorganisms, therefore methods for evaluating hospital surfaces' cleaning gain particular importance. Among these, there are visual inspection, quantitative microbiology, fluorescent markers and adenosine triphosphate (ATP) bioluminescence. The latter seems to provide interesting features, detecting the presence of ATP on surface (as Relative Light Units, RLU), a proxy of organic matter and microbial contamination. Several studies have investigated the effectiveness of this technology; with this research, we aim to summarize the most significant results. METHODS: A systematic review was conducted. The keywords (namely, "ATP", "bioluminescence", "hospital" and "surfaces") were searched in PubMed/MEDLINE and Scopus databases, in order to find relevant data, from January 2000 to October 2014. After the selection, we globally considered 27 articles. RESULTS: Most of the studies were conducted in United Kingdom and in USA. Different threshold RLU benchmark values were identified by analyzed studies. Fourteen of these researches compared the ATP bioluminescence with microbiological methods, 11 identified a significant correlation between the two methods, although poor or not complete for 5. DISCUSSION: ATP bioluminescence is not a standardized methodology: each tool has different benchmark values, not always clearly defined. At the moment, we can say that the technique could be used to assess, in real time, hospital surfaces where cleanliness is required, but not sterility.

Application of ATP-based bioluminescence for bioaerosol quantification: effect of sampling method.

An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60 min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20 mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that that do not affect the bioluminescence signal intensity. Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times. While the ATP-based bioluminescence offers a quick bioaerosol sample analysis method, this works demonstrates that the method output depends on bioaerosol generation and sampling methods, as well as reagent storage.

Hyperlipidemia attenuates vascular endothelial growth factor-induced angiogenesis, impairs cerebral blood flow, and disturbs stroke recovery via decreased pericyte coverage of brain endothelial cells.

OBJECTIVE: Therapeutic angiogenesis aims at the promotion of vascular growth, usually under conditions of atherosclerosis. It was unknown how hyperlipidemia, a risk factor that is closely associated with atherosclerosis of brain vessels in humans, influences vascular endothelial growth factor-induced angiogenesis and stroke recovery. APPROACH AND RESULTS: Wild-type and apolipoprotein-E (ApoE)(-/-) mice were kept on regular or cholesterol-rich diet for mimicking different severities of hyperlipidemia. Mice were treated intracerebroventricularly with recombinant human vascular endothelial growth factor for 21 days (0.02 µg/d) and subsequently subjected to 90-minute middle cerebral artery occlusion followed by 1 or 24 hours of reperfusion. Histochemical, autoradiographic, and regional bioluminescence techniques were used to evaluate effects of blood lipids on postischemic angiogenesis, histopathologic brain injury, cerebral blood flow, protein synthesis and energy state, and pericyte coverage of brain endothelial cells. Hyperlipidemia dose-dependently attenuated vascular endothelial growth factor-induced capillary formation and pericyte coverage of brain endothelial cells, abolishing the improvement of cerebral blood flow during subsequent stroke, resulting in the loss of the metabolic penumbra and increased brain infarction. The enhanced angiogenesis after vascular endothelial growth factor treatment was accompanied by increased expression of the adhesion protein N-cadherin, which mediates endothelial-pericytic interactions, in ischemic brain microvessels of wild-type mice on regular diet that was blunted in wild-type mice on Western diet and ApoE(-/-) mice on either diet. CONCLUSIONS: The compromised vessel formation and hemodynamics question the concept of therapeutic angiogenesis in ischemic stroke where hyperlipidemia is highly prevalent.

Variation in detection limits between bacterial growth phases and precision of an ATP bioluminescence system.

UNLABELLED: To determine the detection limits of the SystemSure Plus, Escherichia coli and Staphylococcus aureus growth curve samples were taken in lag (1 h), log (6 h), stationary (12 h) and death phases (E. coli 144 h, Staph. aureus 72 h). At each time point, the log10 CFU ml(-1) was determined for the dilution where the SystemSure read 0 relative light units (RLU). Average detection limits were E. coli: lag 6·27, log 5·88, stationary 7·45 and death 6·88; Staph. aureus: lag 4·37, log 5·15, stationary 7·88 and death 7·57. Between-run precision was determined with positive control; within-run precision with positive control, lag and log growth for each bacteria. Within-run precision mean RLU (CV): positive control 274 (12%), E. coli lag 1 (63%), log 2173 RLU (19%), Staph. aureus lag 2 (58%) and log 5535 (18%). Between-run precision was 232 (16%). The precision is adequate with most values within the 95% confidence interval. The detection limit varied by 3·51 log10 for Staph. aureus and 1·47 log10 for E. coli. The lowest detection limits were during E. coli log and Staph. aureus lag phases; the highest was during stationary phase. These results suggest that organism identification and growth phase both impact ATP RLU readings. SIGNIFICANCE AND IMPACT OF THE STUDY: Surface hygiene is a critical component of food safety and infection control; increasingly, ATP detection by bioluminescence is used to evaluate surface hygiene and effective cleaning. This is the first study to show that the number of living and potentially infectious bacteria remaining when the device reads zero varies between the different bacterial life cycle phases: lag, log, stationary and death. ATP device users need to be aware of this information to use the devices appropriately.

Air sampling and ATP bioluminescence for quantitative detection of airborne microbes.

Exposure to bioaerosol contamination has detrimental effects on human health. Recent advances in ATP bioluminescence provide more opportunities for the quantitative detection of bioaerosols. Since almost all active organisms can produce ATP, the amount of airborne microbes can be easily measured by detecting ATP-driven bioluminescence. The accurate evaluation of microorganisms mainly relies on following the four key steps: sampling and enrichment of airborne microbes, lysis for ATP extraction, enzymatic reaction, and measurement of luminescence intensity. To enhance the effectiveness of ATP bioluminescence, each step requires innovative strategies and continuous improvement. In this review, we summarized the recent advances in the quantitative detection of airborne microbes based on ATP bioluminescence, which focuses on the advanced strategies for improving sampling devices combined with ATP bioluminescence. Meanwhile, the optimized and innovative strategies for the remaining three key steps of the ATP bioluminescence assay are highlighted. The aim is to reawaken the prosperity of ATP bioluminescence and promote its wider utilization for efficient, real-time, and accurate detection of airborne microbes.

Evaluation of ATP bioluminescence for rapid determination of cleanliness of livestock trailers after a commercial wash.

Pathogens such as porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome (PRRSV), and E. coli are known to spread by contaminated vehicles and equipment. Pork producers have adopted trailer wash policies where each trailer is washed, disinfected, and dried before it can return to a farm. Cleanliness of livestock trailers after washing is determined by visual inspection rather than any objective method. Adenosine triphosphate (ATP) bioluminescence is used in many industries to provide real-time feedback on surface cleanliness through the detection of ATP from organic sources. That same technology may provide trailer wash facilities a way of objectively characterizing a livestock trailer's suitability to return to a farm after washing. Two ATP luminometers (3M Clean-Trace and Neogen AccuPoint) were used to estimate the correlation between ATP bioluminescence readings and aerobic bacterial plate counts (APCs) from sampled surfaces and to determine locations within a livestock trailer that can accurately estimate surface cleanliness. Five locations in livestock trailers were evaluated. Those locations included the nose access door (NAD), back door flush gate, rear side access door (RSAD), belly flush gate (BFG), and belly side access door (BSAD). There was a positive log-log association between the two luminometers (r = 0.59, P < 0.01). Every log unit increase in one unit, resulted in a 0.42 log increase (P < 0.01) in the other unit. ATP can come from bacteria, yeasts, molds, and manure. There was a poor association (r ≥ 0.10, P ≥ 0.02) between APCs and the ATP luminometers. Still, an increase in relative light units (RLUs) resulted in a corresponding increase in colony-forming units. The greatest area of surface contamination measured by APC was the NAD. RLUs were also greater in the NAD compared to the RSAD, the BFG, and the BSAD (P ≤ 0.01). Because APCs and luminometer RLUs provided similar outcomes, statistical process control charts were developed to determine control limits for RLUs. This provides real-time feedback to trailer wash workers in determining cleanliness outcomes for livestock trailers. These data suggest that ATP bioluminescence can be a reliable method to monitor cleaning effectiveness in livestock trailers. Bioluminescence is a monitoring tool that should be used in conjunction with microbial methods to monitor procedures for cleaning and disinfection.

Online monitoring system for qualitative and quantitative analysis of bioaerosols by combined ATP bioluminescence assay with loop-mediated isothermal amplification.

Rapid detection of airborne pathogens is crucial in preventing respiratory infections and allergies. However, technologies aiming to real-time analysis of microorganisms in air remain limited due to the sparse and complex nature of bioaerosols. Here, we introduced an online bioaerosol monitoring system (OBMS) comprised of integrated units including a rotatable stainless-steel sintered filter-based sampler, a lysis unit for extracting adenosine triphosphate (ATP), and a single photon detector-based fluorescence unit. Through optimization of the ATP bioluminescence method and establishment of standard curves between relative luminescence units (RLUs) and ATP as well as microbial concentration, we achieved simultaneous detection of bioaerosols' concentration and activity. Testing OBMS with four bacterial and two fungal aerosols at a sampling flow rate of 10 to 50 L/min revealed an outstanding collection efficiency of 95 % at 30 L/min. A single OBMS measurement takes only 8 min (sampling: 5 min; lysis and detection: 3 min) with detection limits of 3 Pcs/ms photons (2.9 × 103 and 292 CFU/m3 for Staphylococcus aureus and Candida albicans aerosol). In both laboratory and field tests, OBMS detected higher concentrations of bioaerosol compared to the traditional Andersen impactor and liquid biosampler. When combined OBMS with loop-mediated isothermal amplification (LAMP), the bioaerosol can be qualitative and quantitative analyzed within 40 min without the cumbersome procedures of sample pretreatment and DNA extraction. These results offer a high compressive and humidity resistance membrane filtration sampler and validate the potential of OBMS for online measurement of bioaerosol concentration and composition.

A novel phagomagnetic separation-ATP bioluminescence (PhMS-BL) for rapid and sensitive detection of viable Vibrio parahaemolyticus in aquatic product.

Detection of viable Vibrio parahaemolyticus (V. parahaemolyticus) is a major challenge due to its significant risk to food safety and human health. Herein, we developed a phagomagnetic separation-ATP bioluminescence (PhMS-BL) assay based on phage VPHZ6 for rapid and sensitive detection of viable V. parahaemolyticus. Phage as a recognition element was coupled to magnetic beads to capture and enrich V. parahaemolyticus, shortening detection time and improving method sensitivity. The intracellular ATP released by chemical lysis using CTAB was quantified using firefly fluorescein-adenosine triphosphate bioluminescence system to detect viable bacteria. So, PhMS-BL method was able to detect V. parahaemolyticus in a linear range of 2.3 × 102 to 1.3 × 107 CFU mL-1, with a detection limit of 78 CFU mL-1 within 15 min. It is successfully applied to detect V. parahaemolyticus in spiked lake water, lobster tail meat, and clam meat. The developed detection strategy can rapidly and sensitively detect viable V. parahaemolyticus in food matrixes.

Functionalized sampling swabs array-based portable ATP bioluminescence sensor with on-site enrichment and high specificity for live Salmonella detection.

Live food-borne pathogens, featured with rapid proliferative capacity and high pathogenicity, pose an emerging food safety and public health crisis. The high-sensitivity detection of pathogens is particularly imperative yet remains challenging. This work developed a functionalized nylon swab array with enhanced affinity for Salmonella typhimurium (S.T.) for high-specificity ATP bioluminescence-based S.T. detection. In brief, the nylon swabs (NyS) were turned to N-methylation nylon (NyS-OH) by reacting with formaldehyde, and NyS-OH were further converted to NyS-CA by reacting with carboxylic groups of citric acid (CA) and EDC/NHS solution, for altering the NyS surface energy to favor biomodification. The antibody-immobilized nylon swab (MNyS-Ab) was ready for S.T.-specific adsorption. Three prepared MNyS-Ab were installed on a stirrer to form an MNyS-Ab array, allowing for on-site enrichment of S.T. through absorptive extraction. The enriched S.T. was quantified by measuring the bioluminescence of ATP released from cell lysis utilizing a portable ATP bioluminescence sensor. The bioassay demonstrated a detectable range of 102-107 CFU mL-1 with a detection limit (LOD) of 8 CFU/mL within 35 min. The signal of single MNyS-Ab swabs was 500 times stronger than the direct detection of 106 CFU/mL S.T. The MNyS-Ab array exhibited a 100-fold increase in extraction level compared to a single MNyS. This combination of a portable bioluminescent sensor and modified nylon swab array offers a novel strategy for point-of-care testing of live S.T. strains. It holds promise for high-sensitivity measurements of other pathogens and viruses.

Phage-Based Biosensing for Rapid and Specific Detection of Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a major foodborne pathogen. Rapid and specific detection is crucial for controlling staphylococcal food poisoning. This study reported a Staphylococcus phage named LSA2302 showing great potential for applications in the rapid detection of S. aureus. Its biological characteristics were identified, including growth properties and stability under different pH and temperature conditions. The genomic analysis revealed that the phage has no genes associated with pathogenicity or drug resistance. Then, the phage-functionalized magnetic beads (pMB), serving as a biological recognition element, were integrated with ATP bioluminescence assays to establish a biosensing method for S. aureus detection. The pMB enrichment brought high specificity and a tenfold increase in analytical sensitivity during detection. The whole detection process could be completed within 30 min, with a broad linear range of 1 × 104 to 1 × 108 CFU/mL and a limit of detection (LOD) of 2.43 × 103 CFU/mL. After a 2 h pre-cultivation, this method is capable of detecting bacteria as low as 1 CFU/mL. The recoveries of S. aureus in spiked skim milk and chicken samples were 81.07% to 99.17% and 86.98% to 104.62%, respectively. Our results indicated that phage-based biosensing can contribute to the detection of target pathogens in foods.

Comprehensive insights into advances in ambient bioaerosols sampling, analysis and factors influencing bioaerosols composition.

While the study of bioaerosols has a long history, it has garnered heightened interest in the past few years, focusing on both culture-dependent and independent sampling and analysis approaches. Observations have been made regarding the seasonal fluctuations in microbial communities and their connection to particular ambient atmospheric factors. The study of airborne microbial communities is important in public health and atmospheric processes. Nevertheless, the establishment of standardized protocols for evaluating airborne microbial communities and utilizing microbial taxonomy as a means to identify distinct bioaerosols sources and seasonal patterns remains relatively unexplored. This article discusses the challenges and limitations of ambient bioaerosols sampling and analysis, including the lack of standardized methods and the heterogeneity of sources. Future prospects in the field of bioaerosols, including the use of high-throughput sequencing technologies, omics studies, spectroscopy and fluorescence-based monitoring to provide comprehensive incite on metabolic capacity, and activity are also presented. Furthermore, the review highlights the factors that affect bioaerosols composition, including seasonality, atmospheric conditions, and pollution levels. Overall, this review provides a valuable resource for researchers, policymakers, and stakeholders interested in understanding and managing bioaerosols in various environments.