Search

Abstract Microbial flora plays an important role in microorganism-enhanced technology. The pollutant degradation ability and viable counts of these agents are crucial to guarantee their practical application. In this study, an efficient pollutant-degrading microbial flora was screened, its medium components and culture conditions were optimized, and its effect was verified in zeolite trickling filter towers. After a 24 h culture under the optimal conditions, the viable count reached 4.76 × 109 cfu/mL, with the degradation rates of ammonia nitrogen (NH4 +-N), nitrate nitrogen (NO3 −-N), total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) increased to 93.5%, 100%, 68.3%, 32.6%, and 85%, respectively. After optimizing the feeding strategy, the concentration of viable bacteria reached 5.80 × 109 cfu/mL. In the application effect verification experiment, the degradation rates of NH4 +-N, TN, TP, and COD in the experimental group reached 96.69%, 75.18%, 73.82%, and 90.83%, respectively, showing a significant improvement compared to the results of the control group. The main components in the control group were Dokdonella, Brevundimonas, Alishewanella, Rhodobacter, Pseudoxanthomonas, and Thauera, whereas those in the experimental group were Dokdonella, Proteocatella, Rhodobacter, Dechlomonas, and Nitrospira. Proteocatella, Dechlomonas, and Nitrosra, which were unique to the experimental group, are common bacteria used for nitrogen and phosphorus removal. This explains the difference in the sewage treatment capacity between the two groups. This study provides an alternative sewage treatment microbial flora with a reasonable production cost and high degradation efficiency for NH4 +-N, TN, TP, and COD.

Introduction: The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. Methods: We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Results and discussion: Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination. [ABSTRACT FROM AUTHOR]

IntroductionThe microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk.MethodsWe identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells.Results and discussionThree community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Microbial flora plays an important role in microorganism-enhanced technology. The pollutant degradation ability and viable counts of these agents are crucial to guarantee their practical application. In this study, an efficient pollutant-degrading microbial flora was screened, its medium components and culture conditions were optimized, and its effect was verified in zeolite trickling filter towers. After a 24 h culture under the optimal conditions, the viable count reached 4.76 × 109 cfu/mL, with the degradation rates of ammonia nitrogen (NH4+-N), nitrate nitrogen (NO3−-N), total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) increased to 93.5%, 100%, 68.3%, 32.6%, and 85%, respectively. After optimizing the feeding strategy, the concentration of viable bacteria reached 5.80 × 109 cfu/mL. In the application effect verification experiment, the degradation rates of NH4+-N, TN, TP, and COD in the experimental group reached 96.69%, 75.18%, 73.82%, and 90.83%, respectively, showing a significant improvement compared to the results of the control group. The main components in the control group were Dokdonella, Brevundimonas, Alishewanella, Rhodobacter, Pseudoxanthomonas, and Thauera, whereas those in the experimental group were Dokdonella, Proteocatella, Rhodobacter, Dechlomonas, and Nitrospira. Proteocatella, Dechlomonas, and Nitrosra, which were unique to the experimental group, are common bacteria used for nitrogen and phosphorus removal. This explains the difference in the sewage treatment capacity between the two groups. This study provides an alternative sewage treatment microbial flora with a reasonable production cost and high degradation efficiency for NH4+-N, TN, TP, and COD. Keypoints: A mixed-bacteria group with a high degradation rate of NH4+-N, TN, TP, and COD was screened. The viable cell concentration and pollutant degradation efficiency of the screened microbial flora was increased significantly by optimizing its cultivation conditions. Increased pollutant degradation efficiency was verified in a zeolite trickling filter tower and explained by the microbial composition of the screened microbial flora. [ABSTRACT FROM AUTHOR]

Shotgun metagenomic sequencing was used to investigate the diversity of the microbial community of Cheddar cheese ripened over 32 months. The changes in taxa abundance were compared from assembly-based, non-assembly-based, and mOTUs2 sequencing pipelines to delineate the community profile for each age group. Metagenomic assembled genomes (MAGs) passing the quality threshold were obtained for 11 species from 58 samples. Although Lactococcus cremoris and Lacticaseibacillus paracasei were dominant across the shotgun samples, other species were identified using MG-RAST. NMDS analysis of the beta diversity of the microbial community revealed the similarity of the cheeses in older age groups (7 months to 32 months). As expected, the abundance of Lactococcus cremoris consistently decreased over ripening, while the proportion of permeable cells increased. Over the ripening period, the relative abundance of viable Lacticaseibacillus paracasei progressively increased, but at a variable rate among trials. Reads attributed to Siphoviridae and Ascomycota remained below 1% relative abundance. The functional profiles of PMA-treated cheeses differed from those of non-PMA-treated cheeses. Starter rotation was reflected in the single nucleotide variant profiles of Lactococcus cremoris (SNVs of this species using mOTUs2), while the incoming milk was the leading factor in discriminating Lacticaseibacillus paracasei/casei SNV profiles. The relative abundance estimates from Kraken2, non-assembly-based (MG-RAST) and marker gene clusters (mOTUs2) were consistent across age groups for the two dominant taxa. Metagenomics enabled sequence variant analysis below the bacterial species level and functional profiling that may affect the metabolic interactions between subpopulations in cheese during ripening, which could help explain the overall flavour development of cheese. Future work will integrate microbial variants with volatile profiles to associate the development of compounds related to cheese flavour at each ripening stage. [ABSTRACT FROM AUTHOR]

Highlight Research • The shortfin eel elver bicolor bicolor was tested for the safety of Enterobacter sp. JC05, Lactococcus sp. JAL37, and Bacillus sp. PCP1 • The ability of bacterial strains to adhere to epithelial cells of shortfin eel epithelial cells was demonstrated • The bacterial strains' proteolytic, cellulolytic, and lipolytic activities were detected • Oral administration of the bacterial cocktail lowered overall viable bacterial count but did not affect the shortfin eel's intestinal histological characteristics Abstract Aquaculture of the shortfin eel (Anguilla bicolor bicolor) has been plagued by low survival and growth due to the low tolerance to water quality and feed. The microbiota and shape of the fish intestinal tract influence the immune and digestive systems. The use of bacterial probiotics is fascinating to enhance the digestion system. This study aimed to characterize bacterial probiotic candidates' safety and potential probiotic features for shortfin eel (A. bicolor bicolor) aquaculture. The safety, adherence, and enzymatic activity of three bacterial strains (Bacillus sp. PCP1, Lactococcus sp. JAL 37, and Enterobacter sp. JC05) were investigated. An oral application test was performed on shortfin eel (n=880, 15 g) every four days with 0, 3x103, 3x105, and 3x107 CFU/g diet dosages in quadruplicates for two months. At the end of the experiment, total cultivable bacteria and intestinal morphology were assessed. Based on the hemolytic test and intraperitoneal injection, the bacterial strains were considered harmless. In an in vitro investigation, the bacteria attached to shortfin eel intestinal epithelial cells. An agar diffusion method validated the proteolytic, lipolytic, and cellulolytic activities. In vivo feeding tests with the bacterial cocktail lowered the total viable bacteria in the gut while preserving the gut histology. Bacterial strains of the present study are potential probiotic candidates for shortfin (A. bicolor bicolor) aquaculture.

Aquaculture of the shortfin eel (Anguilla bicolor bicolor) has been plagued by low survival and growth due to the low tolerance to water quality and feed. The microbiota and shape of the fish intestinal tract influence the immune and digestive systems. The use of bacterial probiotics is fascinating to enhance the digestion system. This study aimed to characterize bacterial probiotic candidates' safety and potential probiotic features for shortfin eel (A. bicolor bicolor) aquaculture. The safety, adherence, and enzymatic activity of three bacterial strains (Bacillus sp. PCP1, Lactococcus sp. JAL 37, and Enterobacter sp. JC05) were investigated. An oral application test was performed on shortfin eel (n=880, 15 g) every four days with 0, 3x10³, 3x105, and 3x107 CFU/g diet dosages in quadruplicates for two months. At the end of the experiment, total cultivable bacteria and intestinal morphology were assessed. Based on the hemolytic test and intraperitoneal injection, the bacterial strains were considered harmless. In an in vitro investigation, the bacteria attached to shortfin eel intestinal epithelial cells. An agar diffusion method validated the proteolytic, lipolytic, and cellulolytic activities. In vivo feeding tests with the bacterial cocktail lowered the total viable bacteria in the gut while preserving the gut histology. Bacterial strains of the present study are potential probiotic candidates for shortfin (A. bicolor bicolor) aquaculture. [ABSTRACT FROM AUTHOR]

The dark and anoxic environment of ballast tank sediment (BTS) harbors substantial amounts of relic DNA, yet its impact on microbial diversity estimates in BTS management remains poorly understood. This study employed propidium monoazide (PMA) treatment to eliminate relic DNA and used 16S amplicon high-throughput sequencing to characterize both total and viable bacteria. Our findings revealed that relic DNA is abundant in BTS. When removed, it led to variable reductions in species richness, which fluctuated from a 3.15% increase to a 37.52% decrease. Additionally, 6.27%-15.79% of OTUs were absent in the PMA-treated samples. These findings indicate that relic DNA has diverse effects on microbial diversity estimates. Moreover, relic DNA removal altered the relative abundances of a wide range of taxa, thereby facilitating the detection of rare taxa. Furthermore, the absence of relic DNA resulted in an overestimation of co-occurrence network size, complexity, and competitiveness, which could lead to misinterpretations of community assembly processes. In conclusion, our findings indicate that relic DNA obscures microbial diversity estimates and risk assessments in BTS, highlighting the critical need for monitoring viable bacteria in ballast sediment management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota.

Shotgun metagenomic sequencing was used to investigate the diversity of the microbial community of Cheddar cheese ripened over 32 months. The changes in taxa abundance were compared from assembly-based, non-assembly-based, and mOTUs2 sequencing pipelines to delineate the community profile for each age group. Metagenomic assembled genomes (MAGs) passing the quality threshold were obtained for 11 species from 58 samples. Although Lactococcus cremoris and Lacticaseibacillus paracasei were dominant across the shotgun samples, other species were identified using MG-RAST. NMDS analysis of the beta diversity of the microbial community revealed the similarity of the cheeses in older age groups (7 months to 32 months). As expected, the abundance of Lactococcus cremoris consistently decreased over ripening, while the proportion of permeable cells increased. Over the ripening period, the relative abundance of viable Lacticaseibacillus paracasei progressively increased, but at a variable rate among trials. Reads attributed to Siphoviridae and Ascomycota remained below 1% relative abundance. The functional profiles of PMA-treated cheeses differed from those of non-PMA-treated cheeses. Starter rotation was reflected in the single nucleotide variant profiles of Lactococcus cremoris (SNVs of this species using mOTUs2), while the incoming milk was the leading factor in discriminating Lacticaseibacillus paracasei/casei SNV profiles. The relative abundance estimates from Kraken2, non-assembly-based (MG-RAST) and marker gene clusters (mOTUs2) were consistent across age groups for the two dominant taxa. Metagenomics enabled sequence variant analysis below the bacterial species level and functional profiling that may affect the metabolic interactions between subpopulations in cheese during ripening, which could help explain the overall flavour development of cheese. Future work will integrate microbial variants with volatile profiles to associate the development of compounds related to cheese flavour at each ripening stage.

Objective: Bacterial DNA (bactDNA) detection can be used to quickly identify pathogenic bacteria and has been studied on ascitic fluid. We aimed to retrospectively analyze the diagnostic value and applicational prospect of the bactDNA load in spontaneous bacterial peritonitis (SBP). Method: We extracted viable bactDNA from ascitic samples of 250 patients with decompensated cirrhosis collected from October 2019 to April 2021 and detected the bactDNA by droplet digital polymerase chain reaction (ddPCR). We used ascitic samples of a baseline cohort of 191 patients to establish diagnostic thresholds for SBP and analyze the patients' diagnostic performance based on ascites polymorphonuclear (PMN) and clinical manifestation. We performed bactDNA quantification analysis on 13 patients with a PMN less than 250 cells/mm3 but with clinical symptoms. The dynamic changes of the bactDNA load from eight patients (before, during, and after SBP) were analyzed. Results: After the removal of free DNA, the bactDNA detected by ddPCR was generally decreased (1.75 vs. 1.5 log copies/μl, P < 0.001). Compared with the traditional culture and PMN count in the SBP diagnosis, the bactDNA showed that the ddPCR sensitivity was 80.5%, specificity was 95.3%, positive predictive value was 82.5%, and negative predictive value was 94.7%, based on clinical composite criteria. In patients with a PMN less than 250 cells/mm3, the bactDNA load of 13 patients with symptoms was significantly higher than those without symptoms (2.7 vs. 1.7 log copies/μl, P < 0.001). The bactDNA in eight patients had SBP that decreased by 1.6 log copies/μl after 48 h of antibiotic treatment and by 1.0 log copies/μl after 3 days of continued treatment. Conclusion: BactDNA detection can be used to further enhance the diagnostic efficiency of SBP. Therefore, the application of ddPCR assay not only can be used to discriminate and quantify bacteria but also can be used in the clinical assessment for antibiotics treatment. [ABSTRACT FROM AUTHOR]

The microbiota of silage is a key determinant of its quality. Although commercial inoculants are often used to improve silage quality, studies to analyze their impact on the microbiota of preserved forage at farm-scale facilities are scarce. We assessed the diversity of viable bacterial communities of hay (unfermented dry forage) and grass or legume (GL) and corn (C) silage to deepen our knowledge of how inoculant addition drives microbial occurrence patterns on dairy farms. Forage samples were collected from 24 dairy farms over two sampling periods. Samples were analyzed by high-throughput sequencing and quantitative PCR after being treated with propidium monoazide to account for viable cells. We found consistent significant differences between hay and silage community structures across sampling periods. Silage was generally dominated by lactic acid bacteria (LAB), while Pantoea and Sphingomonas were the main co-dominant genera in hay. The GL silage dominated by Pediococcus, Weissella, and Bacillus was phylogenetically different from C silage enriched in Acetobacter. The use of inoculants including Lentilactobacillus buchneri either alone or in combination with Lactiplantibacillus plantarum, Lacticaseibacillus casei, Pediococcus pentosaceus, or Enterococcus faecium did not systematically prevent the occurrence of undesirable bacteria, especially when corn-based, probably because of factors that can mitigate the effect of inoculation on the microbiota. The core Lactobacillales constituted the dominant LAB in silage with up to 96% relative abundance, indicating either the ubiquity of inoculants or the high competitiveness of epiphytes. Silage chemical profiles varied inconsistently with sampling periods and the use of inoculants. Multivariate multi-table analyses allowed the identification of bacterial clusters mainly driven by moisture and magnesium content in hay, while pH, lactic, and fatty acids were the main drivers for silage. Bacterial network analyses showed considerable variations in the topological roles with the use of inoculants. These results may help evaluate the effectiveness of forage management practices implemented on dairy farms and, therefore, are useful for fine-tuning the search for new additives. Such knowledge can be used by forage makers to adjust processing routines to improve the hygienic quality, nutritional potential, and aerobic stability of preserved forage. [ABSTRACT FROM AUTHOR]

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a global disease caused by Mycobacterium tuberculosis (Mtb) and is manifested as a continuum spectrum of infectious states. Both, the most common and clinically asymptomatic latent tuberculosis infection (LTBI), and the symptomatic disease, active tuberculosis (TB), are at opposite ends of the spectrum. Such binary classification is insufficient to describe the existing clinical heterogeneity, which includes incipient and subclinical TB. The absence of clinically TB-related symptoms and the extremely low bacterial burden are features shared by LTBI, incipient and subclinical TB states. In addition, diagnosis relies on cytokine release after antigenic T cell stimulation, yet several studies have shown that a high proportion of individuals with immunoreactivity never developed disease, suggesting that they were no longer infected. LTBI is estimated to affect to approximately one fourth of the human population and, according to WHO data, reactivation of LTBI is the main responsible of TB cases in developed countries. Assuming the drawbacks associated to the current diagnostic tests at this part of the disease spectrum, properly assessing individuals at real risk of developing TB is a major need. Further, it would help to efficiently design preventive treatment. This quest would be achievable if information about bacterial viability during human silent Mtb infection could be determined. Here, we have evaluated the feasibility of new approaches to detect viable bacilli across the full spectrum of TB disease. We focused on methods that specifically can measure host-independent parameters relying on the viability of Mtb either by its direct or indirect detection.

This work aimed to characterize the microbial communities of an anthropogenic soil originating from application of pedotechniques to Vertisols in a Mediterranean environment. Bare soil profiles were sampled at three depths (0–10 cm, 10–30 cm, and 30–50 cm) and compared with the original soil not transformed at the same depths. The anthropogenic soils were characterized by a higher CaCO3 concentration (360–640 g/kg) than control soil (190–200 g/kg), while an opposite trend was registered for clay, where control soil showed a higher concentration (465 g/kg on average) than anthropogenic soil (355 g/kg on average). Organic carbon content was much higher in the untransformed soil. All samples were microbiologically investigated using a combined culture-dependent and -independent approach. Each pedon displayed a generally decreasing level with soil depth for the several microbial groups investigated; in particular, filamentous fungi were below the detection limit at 30–50 cm. To isolate bacteria actively involved in soil particle aggregation, colonies with mucoid appearance were differentiated at the strain level and genetically identified: the major groups were represented by Bacillus and Pseudomonas. MiSeq Illumina analysis identified Actinobacteria and Firmicutes as the main groups. A high microbial variability was found in all the three anthropogenic pedons and the microorganisms constitute a mature community. [ABSTRACT FROM AUTHOR]

The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.

This work aimed to characterize the microbial communities of an anthropogenic soil originating from application of pedotechniques to Vertisols in a Mediterranean environment. Bare soil profiles were sampled at three depths (0–10 cm, 10–30 cm, and 30–50 cm) and compared with the original soil not transformed at the same depths. The anthropogenic soils were characterized by a higher CaCO3 concentration (360–640 g/kg) than control soil (190–200 g/kg), while an opposite trend was registered for clay, where control soil showed a higher concentration (465 g/kg on average) than anthropogenic soil (355 g/kg on average). Organic carbon content was much higher in the untransformed soil. All samples were microbiologically investigated using a combined culture-dependent and -independent approach. Each pedon displayed a generally decreasing level with soil depth for the several microbial groups investigated; in particular, filamentous fungi were below the detection limit at 30–50 cm. To isolate bacteria actively involved in soil particle aggregation, colonies with mucoid appearance were differentiated at the strain level and genetically identified: the major groups were represented by Bacillus and Pseudomonas. MiSeq Illumina analysis identified Actinobacteria and Firmicutes as the main groups. A high microbial variability was found in all the three anthropogenic pedons and the microorganisms constitute a mature community.

The biogeochemical cycling of gold has been proposed from studies focusing on gold particle morphology, surface textures and associated bacteria living on the surface of gold particles. Additionally, it has been suggested that metabolically active bacteria on particles catalyse gold dissolution and gold re-precipitation processes, i.e. fluid–bacterial–mineral interaction within microenvironments surrounding particles. Therefore, the isolation and characterisation of viable bacteria from gold particles can be used as a model to improve the understanding of bacterial–gold interactions. In this study, classical microbiology methods were used to isolate a gold-tolerant bacterium (Acinetobacter sp. SK-43) directly from gold particles. The genome of this isolate contained diverse (laterally acquired) heavy-metal resistance genes and stress tolerance genes, suggesting that gene expression would confer resistance to a wide range of potentially toxic metals that could occur in the surrounding microenvironment. The presence of these genes, along with genes for nutrient cycling under nutrient-limited conditions highlights the genomic capacity of how Acinetobacter sp. SK-43 could survive on gold particles and remain viable. Laboratory experiments demonstrated that this isolate could grow in the presence of soluble gold up to 20 μM (AuCl3) and that >50% of soluble gold was reduced upon exposure. Collectively, these results suggest that Acinetobacter sp. SK-43 (and presumably similar bacteria) could survive the cytotoxic effects of soluble Au from particles undergoing dissolution. This study provides comprehensive insight on the possible bacterial contributions to gold biogeochemical cycling in natural environments. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Existing culture-based instruments for detecting/quantifying proliferating bacteria in suspensions (BACTECTM, BacT/AlertTM, RABITTM etc.) do so based on changes observed in the physical/chemical properties of media (O2/CO2 levels, pH etc.) due to bacterial metabolism. Given the limited metabolic-rate of individual bacterium, they have a “threshold-concentration” of ~107-108CFU/ml, and Times to Detection (TTDs) of 12 hours or longer for low initial loads (24hrs for existing methods).

A method is described to determine viable populations of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The method employs aptamer-magnetic separation combined with resistivity based detection. The bacteria were separated by means of aptamer-functionalized magnetic beads. They were then quantified by measuring their growth kinetics through time-dependent conductivity changes of culture media. The time-course of growth was logged by real-time and contactless measurements that yielded starting concentrations from the duration of lag intervals prior to the log phase of growth. In pure water samples, the linear ranges for measuring E. coli and S. aureus cells are 2.5 × 103–2.5 × 108 CFU·mL−1 and 4.1 × 103–4.1 × 108 CFU·mL−1, respectively. In spiked tap water samples, the lower limits of detection are 2.3 × 104 CFU·mL−1 and 4.0 × 103 CFU·mL−1 for E. coli and S. aureus, with recoveries of 87.0–108.7% and 92.5–105.0%, respectively. The relative standard deviation of these measurements (10.0%) is below that of plate counting method (13.9%). The presence of micro/nanoparticles such as magnetic beads or selenium nanoparticles in the culture media does not interfere, unlike in case of automatted optical density monitoring. The E. coli and S. aureus cells captured on the aptamer-functionalized magnetic beads can be directly tested for their susceptibility to antibiotics. The process of magnetic separation and determination of load burden requires neither bulky, sophisticated equipment nor expensive reagents. [ABSTRACT FROM AUTHOR]

The aim of this randomized, controlled clinical trial was to isolate and identify viable microorganisms in the saliva of study participants that continuously used a stannous and fluoride ion (F/Sn)-containing toothpaste and mouth rinse over a period of three years in comparison to a control group that used stannous ion free preparations (noF/Sn) over the same time period. Each group (F/Sn and noF/Sn) included 16 participants that used the respective oral hygiene products over a 36-month period. Stimulated saliva samples were collected at baseline (T0) and after 36 months (T1) from all participants for microbiological examination. The microbial composition of the samples was analyzed using culture technique, matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry, and 16S rDNA Polymerase Chain Reaction (PCR). There were only minor differences between both groups when comparing the absolute values of viable microbiota and bacterial composition. The treatment with F/Sn led to a slight decrease in disease-associated and a slight increase in health-associated bacteria. It was shown that the use of stannous ions had no negative effects on physiological oral microbiota even after prolonged use. In fact, a stabilizing effect of the oral hygiene products containing stannous ions on the health-associated oral microbiota could be expected.

Aspiration pneumonia can occur in perioperative and older patients, and various oral care methods have been used to prevent it. To validate the effective oral care methods, measuring bacterial counts before and after oral care is necessary. However, isolating and quantifying viable bacteria from those that are inactivated by agents used in oral care is not possible. In this study, we developed a novel method, Delayed real-time PCR (DR-PCR), that can quantify only viable bacteria from mixed samples of viable and dead bacteria. This method takes advantage of the fact that dead bacteria do not grow but viable bacteria do. When the samples were incubated in a liquid medium for 4 hours, the higher the percentage of viable bacteria, the higher the rate of increase in the number of bacteria. This method showed that povidone‑iodine mouthwashing reduced the number of viable bacteria to approximately 1/4 of that before mouthwashing. Although DR-PCR is slightly more time consuming than real-time PCR, it is effective for studying changes in bacterial counts before and after oral care. • We developed a new method to count viable bacteria: Delayed real-time PCR (DR-PCR). • In a mixture of dead and viable bacteria, it quantifies only viable bacteria. • Higher the percent of viable bacteria, the higher the rate of increase in bacteria. • It determines the effectiveness of oral care using disinfectants and antimicrobials. [ABSTRACT FROM AUTHOR]

Background: Spillage of stones in peritoneal cavity is quite common in patients undergoing laparoscopiccholecystectomy. Lost stones may cause wide range of complications postoperatively which reflects theinfective potential of these stones. Objective: The aim of this study was to find out the frequency of viable bacteria in stones retrieved fromthe gallbladder after laparoscopic cholecystectomy & subjecting the stones for culture. Design: Cross-sectional study. Place &Duration: This study was conducted at Surgical Unit 4 of Civil Hospital Karachi,from July 2013 till March 2014. Materials & Metods: 80 cases of cholelithiasis , selected & operated by laparoscopic cholecystectomy wereincluded. During cholecystectomy , stones were collected , intact stones were sent in two separate containersfrom each subject, for microbiological examination. Results: This study included 80 patients, 58 patients(72.5%) were female & 22 patients (27.5%) were male.Out of these 80 patients, 27 patients (33.75%) had positive stone culture, out of these 27 patients , 16 patients(20%) with positive core culture, 11 patients (13.75%) had positive surface culture.The commonest organismwas E.coli in 13 patients (16.3%) followed by Pseudomonas in 3 patients(3.8%), Klebsiella in 3 patients(3.80%), Staphylococcus aureus in 2 patients (2.5%) & Bacteroides fragilis in 1 patient (1.3%). No salmonellaspecies were isolated. All organisms were tested for sensitivity against quinolones,penicillins, cephalosporins,aminoglycosides & carbapenems. Conclusion: This study proves the infective potential of gallstones, as they contain viable bacteria, whichmay cause post operative complications, therefore all spilled stones during surgery should be removed &peritonial cavity should be meticulously cleared.

To ensure the presence of viable Lactobacillus acidophilus in bio‐yoghurts, competitive PCR (cPCR) method was optimised using certain amounts of the recombinant plasmid as a non‐homologous competitor. Results showed that this method was well‐suited to detect and identify approximately 1.3 × 104 cfu/mL of L. acidophilus bacteria. The developed cPCR method was comparable to the cultivation method with a limit of quantification of approximately 104 cfu/g L. acidophilus bacteria. However, the use of MRS‐bile agar medium alone was not enough for an accurate enumeration of viable L. acidophilus bacteria in commercial bio‐yoghurts. We concluded that the combined assay can be used as a more reliable and cost‐effective alternative for the quality assurance of acidophilus yoghurts. [ABSTRACT FROM AUTHOR]

Ancient documents and milestones of human history such as manuscripts and textiles are fragile and during aging undergo chemical, physical, and biological deterioration. Among the different causes of damage, also human intervention plays a role since some restoration strategies proved to be transient and/or they generated further damage. Outdoor monuments undergo deterioration since they are exposed to pollution, weathering, microbial attack (giving rise to undesired pigmentation, discoloration or true dissolution, corrosion, and overall decay), as well as man-made damage (i.e., graffiti). This review article reports the best-fitting strategies used to restore wall paintings, outdoor monuments, textiles, and paper documents to their ancient beauty by employing “soft” biobased approaches such as viable bacteria or suitable enzymes. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes , and the possibility of its direct implementation for routine analyses in the food industry. [ABSTRACT FROM AUTHOR]

• 99.7% of dead bacteria in AGS was shielded by PMA. • Viable bacteria carried 29.0% ∼ 49.0% of iARGs/iMGEs. • 18 of the top 20 dominant genera abundance changes > 1% after PMA treatment. • Multiply i/e/cARGs viable host bacteria were identified and compared. • Viable bacteria and iMGEs explained 64.5% of the tARGs variation. The assessment of antimicrobial resistance (AMR) risk by DNA-based techniques mainly relies on total bacterial DNA. In this case, AMR risk recognition is restricted to the genotype level, lacking crucial phenotypic information, such as the distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in dead and viable bacteria. This limitation hinders the recognition of AMR behavior. Herein, based on propidium monoazide (PMA) shielding method, this work firstly quantified the intracellular ARGs/MGEs in viable and dead bacteria, and the impact of viable bacteria composition on the formation of intracellular/extracellular polymeric substance-related /cell-free ARGs (i/e/cARGs) and MGEs (i/e/cMGEs) in aerobic granular sludge (AGS). The shielding efficiency of PMA against dead bacteria was optimized to be as high as 97.5% when the MLSS of AGS was 2.0 g/L. Under antibiotic stimulation, 29.0% ∼ 49.0% of iARGs/iMGEs were carried by viable bacteria, and the remaining proportion were carried by dead bacteria. 18 out of the top 20 dominant genera showed a change in abundance by more than 1% after PMA treatment. 29 viable hosts were identified to associate with 52 iARGs, of which 28 and 15 hosts were also linked to 40 eARGs and 26 cARGs. Also, partial least-squares path model and variance partitioning analysis disclosed that viable bacteria and i/e/cMGEs had a positive effect on i/e/cARGs, with both contributing as much as 64.5% to the total ARGs enrichment. These results better visualized the AMR risk carried by viable bacteria and the categories of viable hosts. This work provides a novel insight into analyzing the actual AMR risk and viable hosts, helping to the reduction and control of AMR in wastewater treatment plants. [Display omitted] [ABSTRACT FROM AUTHOR]

Staphylococcus aureus may cause infections in humans from mild skin disorders to lethal pneumonia. Rapid and accurate monitoring of viable S. aureus is essential to characterize human exposure. This study evaluated quantitative PCR ( qPCR) with propidium monoazide ( PMA) to quantify S. aureus. The results showed comparable S. aureus counts between exclusively live cells and mixtures of live/dead cells by qPCR with 1.5 or 2.3 μg/ mL PMA ( P>.05), illustrating the ability of PMA- qPCR to detect DNA exclusively from viable cells. Moreover, qPCR with 1.5 or 2.3 μg/ mL PMA performed optimally with linearity over 103-108 CFU/ mL ( R2≥0.9), whereas qPCR with 10, 23 or 46 μg/ mL PMA significantly underestimated viable counts. Staphylococcus aureus and total viable bacteria were further determined with PMA- qPCR (1.5 μg/ mL) from 48 samples from a public library and two university dormitories and four from outside. Viable bacteria averaged 1.9×104 cells/m3, and S. aureus were detected in 22 (42%) samples with a mean of 4.4×103 cells/m3. The number of S. aureus and viable bacteria were positively correlated (r=.61, P<.005), and percentages of S. aureus relative to viable bacteria averaged 12-44%. The results of field samples suggest that PMA- qPCR can be used to quantify viable S. aureus cells. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Drinking water disinfection plays a critical role in protecting humans from waterborne pathogens. Ferrate(VI) (FeO ) has also been proposed as a disinfectant. This is the first study investigating the bacterial microbiomes of ferrate(VI)-treated water compared to chlorinated water. Tested water was collected after sand filtration and before disinfection from a drinking water treatment plant at Jiaxing, Zhejiang Province, China. A culture-independent method utilizing propidium monoazide was used with quantitative polymerase chain reaction and pyrosequencing of 16S rRNA genes to distinguish between the viable and nonviable bacterial populations. The operational taxonomic units and α-diversity indexes of the live bacterial phylotypes in the samples were determined. Viable bacteria remained in all samples following chlorination or ferrate treatment. However, the genera Vibrio, Salmonella, Shigella, Escherichia, Campylobacter, Yersinia, Plesiomonas, Legionella, and Helicobacter, which contain important human pathogens, were not present among the 25 dominant genera seen in these samples. The profiles of the bacteria remaining after treatment with either chlorine or ferrate differed. The ferrate-treated samples showed a reduced percent relative abundance of operational taxonomic units of the class Alphaproteobacteria within the total remaining viable bacteria. The genera Flavobacterium and Duganella were relatively resistant to treatment by either chlorine or ferrate(VI). At the highest doses of chlorine and ferrate(VI), the genus Sphingobium represented a greater percentage of live bacteria in the chlorinated sample than in the ferrate(VI)-treated sample. The results suggest that ferrate(VI) and chlorine could inactivate slightly different sets of bacteria and could have different mechanisms of bacterial inactivation. [ABSTRACT FROM AUTHOR]

Existing culture-based instruments for detecting/quantifying proliferating bacteria in suspensions (BACTECTM, BacT/AlertTM, RABITTM etc.) do so based on changes observed in the physical/chemical properties of media (O2/CO2 levels, pH etc.) due to bacterial metabolism. Given the limited metabolic-rate of individual bacterium, they have a “threshold-concentration” of ~107-108CFU/ml, and Times to Detection (TTDs) of 12 hours or longer for low initial loads (<100CFU/ml). We recently developed a method that tracks microbial proliferation in suspensions by monitoring the degree of cell polarization of live microorganisms. In the presence of an AC electric field, there occurs a build-up of charge at the microbial membrane, causing them to act like capacitors. As microorganisms multiply, there occurs a corresponding increase in charges stored in the suspension (“bulk-capacitance”), and this increase in bulk-capacitance serves as our “signature” for presence of live microorganisms. In this study, we explain the theory underlying our approach, establish its applicability to a variety of microorganisms, showing that the “Threshold-Concentration” (nT) for detection is ~103-104CFU/ml, and TTDs are a function of the initial-load(n0) and doubling-time(tD) of the microorganism TTD=1.443*tD*ln(nT/n0) and show that the method can be adapted to obtain the “Most Probable Number” (MPN) of coliforms within 6hrs (vs. >24hrs for existing methods). [ABSTRACT FROM AUTHOR]

Viable bacterial bioaerosols cause infections, inflammation and allergic responses in humans and require analytical techniques for exposure assessment. Conventional culture assays often underestimate bioaerosol burdens. This study assessed quantitative PCR (qPCR)-based method considering the effects of DNA dyes (propidium monoazide (PMA) and ethidium monoazide (EMA)), cell densities and dead cells. By testing PMA and EMA at 0.2–46 μg/mL with cultivated air samples, qPCR with PMA of 1.5 μg/mL was revealed optimal for exclusively quantifying total counts of viable bacteria with a great linearity (r=0.99) over 7 orders of magnitude (4–10 log CFU/mL) and no interference by heat-inactivated cells (8 log CFU/mL). Resulting gene copies were properly transformed to bacterial numbers by constructed calibration curves (R 2 =0.96–0.97). Total, viable and culturable bacteria in the air of various indoor and outdoor environments were further quantified and a descending order of total counts>viable counts>culturable counts was observed regardless of sampling location, highlighting the applicability of PMA-qPCR in field aerobiology. Besides, by respectively dividing the concentration of viable and culturable cells by that of total cells, there were 13.7–71.7% of airborne bacteria detected viable compared to 0.3–1.2% of cells as culturable, illustrating high quantities of viable but not culturable bacteria in field air. [ABSTRACT FROM AUTHOR]

The plate counting (PC) method is commonly used to determine the total bacterial count (TBC), which includes all viable bacterial cells displaying growth activity. However, this method is time-consuming and may not effectively identify all actively growing bacteria, and it remains largely unknown whether alternative methods can rapidly and universally quantify the TBC of growth-active bacteria. In this study, we investigated the use of fluorescent d -amino acid (FDAA)-based labeling, which universally targets viable bacteria, in conjunction with typical bacterial species found in milk. Furthermore, we established an antibiotic cocktail to impede cell division during the FDAA labeling process. We optimized the FDAA labeling and cell division inhibition conditions to integrate with flow cytometry (FCM), enabling the rapid quantification of viable bacteria with growth activity. Ultimately, we successfully demonstrated the universal quantification of viable bacteria in real milk samples with a TBC higher than 8.9 × 104 cells/mL within a timeframe of 2.5 h using the FDAA-FCM method. These findings suggest that the FDAA-FCM method holds great promise for quantifying TBC in complex biological samples, offering a rapid and universally applicable approach. • A novel method was built to rapidly and exclusively quantify viable bacteria in milk. • FDAA was used to exclusively identify viable bacteria with growth activity. • Antibiotics were used to maintain the total bacteria count in FDAA labeling process. • FDAA-FCM method rapidly quantifies all viable bacteria with growth activity. • FDAA-FCM identifies all viable bacteria and can amend the plate counting method. [ABSTRACT FROM AUTHOR]

Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis. • CRISPR-leveraged biosensors for POC testing of pathogenic biosafety. • Integration of bioaffinities broadens target scope of CRISPR-based biosensing. • CRISPR-enabled pathogenic biosafety analysis was highlighted. • Challenges of CRISPR-based biosensing in pathogenic biosafety were discussed. [ABSTRACT FROM AUTHOR]

Viable bacteria on water-insoluble airborne particles were detected in the urban atmosphere of Kumamoto (134°45′E, 32°28′N), Japan, in autumn 2008. Airborne particles were collected onto film-covered Cu meshes under clear weather conditions. The samples were stained by fluorescent stains, and then viewed and photographed with an epifluorescent microscope. Non-biological and bacterial parts in particles larger than 0.8 μm were distinguished by their morphologies, fluorescent colors and fluorescent intensities. Bacterial viable statuses were discriminated according to cell membrane damage. In total, 2681 particles were investigated and it was found that 78 airborne particles were associated with bacteria. Viable bacteria were identified on 48 particles. A few particles carried multiple viable bacteria. These results provide the evidence that airborne particles act as carriers of viable bacteria in the atmosphere.

In shallow sublittoral rocky habitats of the Southeast Pacific, two conspicuous ecological systems exist; kelp beds dominated by large Laminariales algae, and barren grounds dominated by crustose coralline algae and sea urchins. The aim of this study was to examine the successional development of micro-periphyton communities in both ecological systems using a colonization experiment conducted in Northern Chile. In both ecological systems, we installed replicated ceramic plates at 10 m depth and samples were taken after 1, 2, 4, 6, 8 and 14 days of exposure. Bacteria, diatoms, protozoans and small eukaryotes were identified, quantified and analysed. The succession of micro-periphyton communities in both ecological systems followed a common pattern consisting of a net accumulation of functional groups and taxa over time; however, the total density of all groups was significantly higher in the kelp beds. In addition, the community structure of the developing micro-periphytons was different and specific for each ecological system. Although previous studies have suggested that kelp beds and barren grounds are capable of switching from one state to the other without substantial changes in biodiversity, our results show that each of these ecological systems promotes its own successional development, suggesting that they are unique, self-organized entities. This study is the first step towards an understanding of these ecological systems operating independently at this scale of organization. [ABSTRACT FROM AUTHOR]

The objectives of this study were (i) to assess the determinants that affect concentrations of the bacterial cell wall components 3-hydroxy fatty acids (3- OH FAs) and muramic acid and of total viable bacteria and actinomycetes in house dust; and (ii) to examine the seasonal variation and reproducibility of these bacterial cell wall components in house dust. A number of lifestyle and environmental factors, mostly not consistent for different bacterial measures but commonly including the type of dwelling and farming (number of livestock), explained up to 37% of the variation of the bacterial concentrations in 212 homes in Eastern Finland. The reproducibility of 3- OH FAs and muramic acid measurements in house dust were studied in five urban homes and were found to be generally high ( ICC 74-84%). Temporal variation observed in repeated sampling of the same home throughout a year was more pronounced for 3- OH FAs determinations ( ICC 22%) than for muramic acid ( ICC 55-66%). We conclude that determinants vary largely for different types of bacterial measurements in house dust; the measured parameters represent different aspects of the bacterial content indoors. More than one sample is needed to describe bacterial concentrations in house dust in the home environment due to large temporal variation. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Actinobacillus (A.) pleuropneumoniae is a causative agent of porcine pleuropneumonia, which has been reported in most countries. A genomic expression library of A. pleuropneumonia serotype 7 was constructed to identify potential vaccine candidates. Plasmid DNA from the sub-libraries (L1 to L10) was used to immunize BALB/c mice. Then, 2 weeks after the final immunization, the groups of mice were challenged with A. pleuropneumonia serotype 1 (1?109 cfu). Mice immunized with one of the sublibraries (L3) had a higher level of antibodies and IFN-γ production than others. This sub-library (L3) resulted in a significant reduction in the amount of A. pleuropneumoniae recovered from mouse lungs and the survival rate of the L3 group was the most highest. Our study demonstrates that a genome expression library of A. pleuropneumoniae serotype 7 offers a novel approach for screening possible cross-serotype protection vaccine candidates against A. pleuropneumoniae serotype 1 infection. [ABSTRACT FROM AUTHOR]

Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the associated public health risk. Discrepancies between results obtained by molecular and conventional culture methods stem from the difference in target, namely nucleic acids instead of actively growing microorganisms. Nucleic acids constitute 5% to 15% of the dry weight of all living cells and are relatively stable, even after cell death, so they may be present in a food matrix after the foodborne microorganisms have been inactivated. Therefore, interpretation of the public health significance of positive results generated by nucleic acid detection methods warrants some additional consideration. This review discusses the stability of nucleic acids in general and highlights the persistence of microbial nucleic acids after diverse food-processing techniques based on data from the scientific literature. Considerable amounts of DNA and RNA (intact or fragmented) persist after inactivation of bacteria and viruses by most of the commonly applied treatments in the food industry. An overview of the existing adaptations for molecular assays to cope with these problems is provided, including large fragment amplification, flotation, (enzymatic) pretreatment, and various binding assays. Finally, the negligible risks of ingesting free microbial nucleic acids are discussed and this review ends with the future perspectives of molecular methods such as next-generation sequencing in diagnostic and source attribution food microbiology. [ABSTRACT FROM AUTHOR]

Citrus canker [caused by Xanthomonas citri subsp. citri ( Xcc)] can cause yield loss of susceptible citrus and result in trade restrictions of fresh fruit. For both regulatory purposes and epidemiological studies, accurate detection and quantification of viable inoculum are critical. Two accepted methods used to detect and quantify Xcc are injection-infiltration bioassay and culture, but these two methods have not been directly compared using field-obtained samples. The two methods were compared using washates of lesions taken from fruit, leaves and shoots in a commercial orchard in Florida in 2009-2010 and 2010-2011, with bioassay being the assumed standard. Despite some misclassifications, true positives (sensitivity) and true negatives (specificity) were the dominant classes using culture. False positives for lesions from shoots ranged from 13.1 to 21.4% in 2009-2010 and 2010-2011, respectively, and false positives for lesions from fruit and leaves ranged from 4.3 to 15.7%, in the two seasons, respectively. The false positive rate for culture compared with injection-infiltration bioassay was highest (0.16-0.55), due to more frequent recovery of Xcc by culture at ≤103 colony-forming units ( CFU) Xcc per ml. The false negative rate was consistently lower (0.02-0.21), confirming that in only a few cases did culture fail to detect Xcc when it was present. The area under the curve for receiver operator characteristic analysis ranged from 0.80 to 0.97, confirming that culture provided an accurate diagnosis in most cases. There was a higher frequency of lesions from shoots with a CFU ≤103 Xcc compared with lesions from fruit or leaves, making culture more effective at detecting these. The data demonstrate that culture is a reliable way to detect and quantify Xcc compared with injection-infiltration bioassay, particularly when the CFU is ≤103 Xcc per ml. [ABSTRACT FROM AUTHOR]

Abstract: Helicobacter (H.) suis colonizes the gastric mucosa of pigs world-wide and is the most prevalent non-Helicobacter pylori Helicobacter species in humans. This agent might be transmitted to humans by manipulation or consumption of contaminated pork. H. suis is a very fastidious micro-organism and is extremely difficult to isolate. Therefore, we developed a non-culture dependent, quantitative detection method allowing differentiation of viable from dead H. suis bacteria in pork. This was established by a combination of ethidium bromide monoazide (EMA) treatment and real-time (RT)-PCR. This EMA RT-PCR was applied to 50 retail pork samples. In two samples, viable H. suis bacteria were detected. Sequence analysis of the obtained PCR products confirmed the presence of H. suis DNA. Viable H. suis bacteria persisted for at least 48h in experimentally contaminated pork. In conclusion, consumption of contaminated pork may constitute a new route of transmission for H. suis infections in humans. [Copyright &y& Elsevier]

Tuberculosis (TB) is a global disease caused by Mycobacterium tuberculosis ( Mtb ) and is manifested as a continuum spectrum of infectious states. Both, the most common and clinically asymptomatic latent tuberculosis infection (LTBI), and the symptomatic disease, active tuberculosis (TB), are at opposite ends of the spectrum. Such binary classification is insufficient to describe the existing clinical heterogeneity, which includes incipient and subclinical TB. The absence of clinically TB-related symptoms and the extremely low bacterial burden are features shared by LTBI, incipient and subclinical TB states. In addition, diagnosis relies on cytokine release after antigenic T cell stimulation, yet several studies have shown that a high proportion of individuals with immunoreactivity never developed disease, suggesting that they were no longer infected. LTBI is estimated to affect to approximately one fourth of the human population and, according to WHO data, reactivation of LTBI is the main responsible of TB cases in developed countries. Assuming the drawbacks associated to the current diagnostic tests at this part of the disease spectrum, properly assessing individuals at real risk of developing TB is a major need. Further, it would help to efficiently design preventive treatment. This quest would be achievable if information about bacterial viability during human silent Mtb infection could be determined. Here, we have evaluated the feasibility of new approaches to detect viable bacilli across the full spectrum of TB disease. We focused on methods that specifically can measure host-independent parameters relying on the viability of Mtb either by its direct or indirect detection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alebouyeh, Weinrick, Achkar, García and Prados-Rosales.)