Microbial source tracking and identification of fecal contamination patterns in saltwater estuaries between base- and storm-flow events using dual genome-based approaches.

Fecal contamination from natural and anthropogenic sources poses significant threats to saltwater estuaries, particularly after storms or heavy rainfall. Monitoring fecal contamination is essential for protecting these vulnerable ecosystems having important ecological and economic values. In this study, we investigated the abundance, sources, and potential causes of fecal contamination at three marine and seven freshwater stations across Vaughn Bay (WA, USA), a shellfish growing district, during base- and storm-flow events. Additionally, we evaluated the performance of fecal indicator bacteria (FIB) quantification, optical brightener assessment, and qPCR analysis for fecal contamination quantification. We compared the effectiveness of qPCR-based microbial source tracking (MST), which targeted a broad range of hosts including, such as humans, birds, cows, horses, ruminants, dogs, and pigs, with sequencing-based MST in identifying fecal contamination sources. Both MST analysis approaches identified birds and humans as the primary sources of fecal contamination. For marine water stations, freshwater creeks VBU001, VBU002, and VB047, along with drain VB007, were identified as the main sources of human-derived fecal contamination in Vaughn Bay, based on Kendall's tau analysis (τ: 0.58-0.97). This information indicates that the septic systems in the catchment areas of these creeks and drains require further investigation to achieve effective fecal contamination control. Optical brightener, FIB enumeration and qPCR quantification results were generally higher during storm-flow events, although they showed poor correlation with each other (Pearson r < 0.40), likely due to physiological and phylogenetic differences among the target organisms of these methods. However, the sequencing-based method faces challenges in precise quantitative identification of differences in fecal contamination between base- and storm-flow events. Due to its high-throughput and cost-effectiveness, we recommend using sequencing-based analysis for large-scale identification of the primary sources of fecal contamination in water environments, followed by targeted qPCR quantification of MST markers for more precise assessments.

Improvement in Microbiota Recovery Using Cas-9 Digestion of Manuka Plastid and Mitochondrial DNA.

Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (manuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.

What is the Difference between Conventional Drinking Water, Potable Reuse Water, and Nonpotable Reuse Water? A Microbiome Perspective.

As water reuse applications expand, there is a need for more comprehensive means to assess water quality. Microbiome analysis could provide the ability to supplement fecal indicators and pathogen profiling toward defining a "healthy" drinking water microbiota while also providing insight into the impact of treatment and distribution. Here, we utilized 16S rRNA gene amplicon sequencing to identify signature features in the composition of microbiota across a wide spectrum of water types (potable conventional, potable reuse, and nonpotable reuse). A clear distinction was found in the composition of microbiota as a function of intended water use (e.g., potable vs nonpotable) across a very broad range of U.S. water systems at both the point of compliance (Betadisper p > 0.01; ANOSIM p < 0.01, r-stat = 0.71) and point of use (Betadisper p > 0.01; ANOSIM p < 0.01, r-stat = 0.41). Core and discriminatory analysis further served in identifying distinct differences between potable and nonpotable water microbiomes. Taxa were identified at both the phylum (Desulfobacterota, Patescibacteria, and Myxococcota) and genus (Aeromonas and NS11.12_marine_group) levels that effectively discriminated between potable and nonpotable waters, with the most discriminatory taxa being core/abundant in nonpotable waters (with few exceptions, such as Ralstonia being abundant in potable conventional waters). The approach and findings open the door to the possibility of microbial community signature profiling as a water quality monitoring approach for assessing efficacy of treatments and suitability of water for intended use/reuse application.

Vaginal microbiota stability over 18 months in young student women in France.

PURPOSE: Non-optimal vaginal microbiota lacking lactobacilli and comprising a wide array of anaerobic bacteria, typified by community state type (CST) IV, have been associated with adverse gynecological and pregnancy outcomes. Here, we investigate the stability of the vaginal microbiota sampled every 6 months over 18 months and how samples distantly collected combined with exposures could provide insight on future microbiota compositional changes. METHODS: Vaginal microbiota dynamics were analyzed in 241 female students aged 18-24 years and negative for Chlamydia trachomatis and Neisseria gonorrhoeae. The vaginal microbiota was characterized using 16S rRNA gene amplicon sequencing and assigned to CSTs. Vaginal microbiota longitudinal profiles were determined through hierarchical clustering. RESULTS: At baseline, 11.2% of participants had a CST IV, 40.5% a CST I (Lactobacillus crispatus-dominated), and 38.1% a CST III (Lactobacillus iners-dominated). A total of 345 CST transitions were observed over the study period. Pain during sexual intercourse was associated with a higher probability of transition from CST III to CST IV, while self-reported yeast infection was associated with a higher probability of transition from CST IV to CST I. Over the study period, 32.0% participants displayed a stable CST trajectory. Composition of the vaginal microbiota of a single sample predicted with good accuracy the CST trajectory over the following 18 months. CONCLUSION: Vaginal longitudinal CST patterns over 18 months could be clustered into three main groups of trajectories. Performing molecular characterization at a single time point could contribute to improved preventive care and optimization of young women's reproductive and sexual health. CLINICALTRIALS: gov Identifier: NCT02904811. Registration date: September 19, 2016.

Associations of Neonatal Dairy Calf Faecal Microbiota with Inflammatory Markers and Future Performance.

After birth, the immune system is challenged by numerous elements of the extrauterine environment, reflected in fluctuations of inflammatory markers. The concentrations of these markers in the first month of life are associated with the future performance of dairy youngstock. It is thought that bacterial genera colonizing the calf intestinal tract can cause inflammation and thus affect their host's performance via immunomodulation. This study explored how the faecal microbiota of newborn dairy calves were related to inflammatory markers during the first three weeks of life, and if the abundance of specific genera was associated with first-lactation performance. Ninety-five female Holstein calves were studied. Once a week, serum and faecal samples were collected, serum concentrations of serum amyloid A, haptoglobin, tumour necrosis factor-α, and interleukin-6 were measured, and faecal microbiota composition was examined by 16S rRNA gene amplicon sequencing. Faecal Gallibacterium abundance in the first week of age and Collinsella abundance in the second week were negatively associated with inflammatory response as well as with calving-conception interval. Peptostreptococcus abundance in the second week of life was positively associated with inflammatory response and calving-conception interval, and negatively with average daily weight gain. In the third week, Dorea abundance was positively, Bilophila abundance was negatively associated with inflammatory response, and both genera were negatively associated with age at first calving. These bacterial genera may be able to influence the inflammatory response and through this, possibly the future performance of the dairy heifer. Deciphering such microbiota-host interactions can help improve calf management to benefit production and welfare.

Characterization of the gastrointestinal microbiome of the Syrian hamster (Mesocricetus auratus) and comparison to data from mice.

Syrian hamsters (Mesocricetus auratus) have been increasingly used as rodent models in recent years, especially for SARS-CoV-2 since the pandemic. However, the physiology of this animal model is not yet well-understood, even less when considering the digestive tract. Generally, the gastrointestinal microbiome influences the immune system, drug metabolism, and vaccination efficacy. However, a detailed understanding of the gastrointestinal microbiome of hamsters is missing. Therefore, we analyzed 10 healthy 11-week-old RjHan:AURA hamsters fed a pelleted standard diet. Their gastrointestinal content was sampled (i.e., forestomach, glandular stomach, ileum, cecum, and colon) and analyzed using 16S rRNA gene amplicon sequencing. Results displayed a distinct difference in the bacterial community before and after the cecum, possibly due to the available nutrients and digestive functions. Next, we compared hamsters with the literature data of young-adult C57BL/6J mice, another important animal model. We sampled the same gastrointestinal regions and analyzed the differences in the microbiome between both rodents. Surprisingly, we found strong differences in their specific gastrointestinal bacterial communities. For instance, Lactobacillaceae were more abundant in hamsters' forestomach and ileum, while Muribaculaceae dominated in the mouse forestomach and ileum. Similarly, in mouse cecum and colon, Muribaculaceae were dominant, while in hamsters, Lachnospiraceae and Erysipelotrichaceae dominated the bacterial community. Molecular strains of Muribaculaceae in both rodent species displayed some species specificity. This comparison allows a better understanding of the suitability of the Syrian hamster as an animal model, especially regarding its comparability to other rodent models. Thereby, this work contributes to the characterization of the hamster model and allows better experimental planning.

The effects of different lung parts, age, and batches on the lung microbiota of healthy rats.

BACKGROUND: Rat models are valuable tools to study the lung microbiota in diseases. Yet the impacts of different lung parts, young and mature adult stages, and the different batches of the same conditions on the healthy rat lung microbiome have not been investigated. METHODS: The rat lung microbiome was analyzed to clarify the lung part-dependent and age-dependent differences and to evaluate the effects of several 'batch environmental factors' on normal rats, after eliminating potential contamination. RESULTS: The results showed that the contamination could be identified and excluded. The lung microbiome from left and right lung parts was very similar so one representative part could be used in the microbiome study. There were significantly different lung microbial communities between the young and mature adult groups, and also between the different feeding batches groups of the same repetitive feeding conditions, but a common lung microbiota characterized by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria as the most dominant phyla were present in all adult rats. It indicated that the experiment under the same condition of the same rats batch was needed to compare the difference in the lung microbiota and repeated experiments were necessary to confirm the results. CONCLUSION: These data represented that the lung bacterial communities were dynamic and rapidly susceptible to environmental influence, clustered strongly by age or different feeding batches but similar in the different lung tissue parts. This study improved the basic understanding of the potential effects on the lung microbiome of healthy rats.

Exploring the Microbial Diversity and Composition of Three Cigar Product Categories.

Cigars and cigarillos are emerging as popular tobacco alternatives to cigarettes. However, these products may be equally harmful to human health than cigarettes and are associated with similar adverse health effects. We used 16S rRNA gene amplicon sequencing to extensively characterize the microbial diversity and investigate differences in microbial composition across 23 different products representing three different cigar product categories: filtered cigar, cigarillo, and large cigar. High throughput sequencing of the V4 hypervariable region of the 16 s rRNA gene revealed 2124 Operational Taxonomic Units (OTUs). Our findings showed that the three categories of cigars differed significantly in observed richness and Shannon diversity, with filtered cigars exhibiting lower diversity compared to large cigars and cigarillos. We also found a shared and unique microbiota among different product types. Firmicutes was the most abundant phylum in all product categories, followed by Actinobacteria. Among the 16 genera shared across all product types were Bacillus, Staphylococcus, Pseudomonas, and Pantoea. Nine genera were exclusively shared by large cigars and cigarillos and an additional thirteen genera were exclusive to filtered cigars. Analysis of individual cigar products showed consistent microbial composition across replicates for most large cigars and cigarillos while filtered cigars showed more inter-product variability. These findings provide important insights into the microbial diversity of the different cigar product types.

Effect of a Higher-Protein Nut versus Higher-Carbohydrate Cereal Enriched Diet on the Gut Microbiomes of Chinese Participants with Overweight and Normoglycaemia or Prediabetes in the Tu Ora Study.

Global increases in metabolic disorders such as type 2 diabetes (T2D), especially within Asian populations, highlight the need for novel approaches to dietary intervention. The Tu Ora study previously evaluated the effects on metabolic health of including a nut product into the diet of a New Zealand cohort of Chinese participants with overweight and normoglycaemia or prediabetes through a 12-week randomised, parallel-group clinical trial. In this current study, we compared the impact of this higher-protein nut bar (HP-NB) versus a higher-carbohydrate cereal bar (HC-CB) on the faecal microbiome by employing both 16S rRNA gene amplicon and shotgun metagenomic sequencing of pre- and post-intervention pairs from 84 participants. Despite the higher fibre, protein, and unsaturated fat content of nuts, there was little difference between dietary groups in gut microbiome composition or functional potential, with the bacterial phylum Firmicutes dominating irrespective of diet. The lack of observed change suggests the dietary impact of the bars may have been insufficient to affect the gut microbiome. Manipulating the interplay between the diet, microbiome, and metabolic health may require a more substantial and/or prolonged dietary perturbation to generate an impactful modification of the gut ecosystem and its functional potential to aid in T2D risk reduction.

Epiphytic Bacterial Community Analysis of Ulva prolifera in Garorim and Muan Bays, Republic of Korea.

The bacterial communities related to seaweed can vary considerably across different locations, and these variations influence the seaweed's nutrition, growth, and development. To study this further, we evaluated the bacteria found on the green marine seaweed Ulva prolifera from Garorim Bay and Muan Bay, two key locations on Republic of Korea's west coast. Our analysis found notable differences in the bacterial communities between the two locations. Garorim Bay hosted a more diverse bacterial population, with the highest number of ASVs (871) compared to Muan Bay's 156 ASVs. In Muan Bay, more than 50% of the bacterial community was dominated by Pseudomonadota. On the other hand, Garorim Bay had a more balanced distribution between Bacteroidota and Pseudomonadota (37% and 35.5%, respectively). Additionally, Cyanobacteria, particularly Cyanothece aeruginosa, were found in significant numbers in Garorim Bay, making up 8% of the community. Mineral analysis indicated that Garorim Bay had higher levels of S, Na, Mg, Ca, and Fe. Function-wise, both locations exhibited bacterial enrichment in amino acid production, nucleosides, and nucleotide pathways. In conclusion, this study broadens our understanding of the bacterial communities associated with Ulva prolifera in Korean waters and provides a foundation for future research on the relationships between U. prolifera and its bacteria.

Faecal bacterial communities differ amongst discrete foraging populations of dugongs along the east Australian coast.

Gut bacterial communities play a vital role in a host's digestion and fermentation of complex carbohydrates, absorption of nutrients, and energy harvest/storage. Dugongs are obligate seagrass grazers with an expanded hindgut and associated microbiome. Here, we characterised and compared the faecal bacterial communities of dugongs from genetically distinct populations along the east coast of Australia, between subtropical Moreton Bay and tropical Cleveland Bay. Amplicon sequencing of fresh dugong faecal samples (n=47) revealed Firmicutes (62%) dominating the faecal bacterial communities across all populations. Several bacterial genera (Bacteroides, Clostridium sensu stricto 1, Blautia and Polaribacter) were detected in samples from all locations, suggesting their importance in seagrass digestion. Principal coordinate analysis showed the three southern-most dugong populations having different faecal bacterial community compositions from northern populations. The relative abundances of the genera Clostridium sensu stricto 13 and dgA-11 gut group were higher, but Bacteroides was lower, in the southern dugong populations, compared to the northern populations, suggesting potential adaptive changes associated with location. This study contributes to our knowledge of the faecal bacterial communities of dugongs inhabiting Australian coastal waters. Future studies of diet selection in relation to seagrass availability throughout the dugong's range will help to advance our understanding of the roles that seagrass species may play in affecting the dugong's faecal bacterial community composition.

Impact of rumen cannulation surgery on rumen microbiota composition in Hanwoo steers.

Rumen cannulation is a surgical technique used to collect rumen contents from ruminants. However, rumen cannulation surgery may potentially impact the composition of the rumen microbiota. This study aimed to examine the longitudinal alterations in the rumen microbiota composition of Hanwoo steers after cannulation surgery. In this study, eight Hanwoo steers were used; four steers underwent rumen cannulation surgery (cannulation group), while the remaining four were left intact (control group). Rumen samples were collected from all eight steers using the stomach tubing method on the day before surgery (day 0) and on postoperative days 1, 4, 7, 10, 14, 17, 21, 24, and 28, resulting in 80 samples (10 timepoints × 8 animals). The microbiota of all 80 samples were analyzed using 16S rRNA gene amplicon sequencing with Quantitative Insights into Microbial Ecology version 2 (QIIME2). There were no significant differences (p > 0.05) in all major phyla and most major genera representing at least 0.5% of total sequences across all 80 samples between the control and cannulation groups on the preoperative and postoperative days. However, while the alpha diversity indices did not differ (p > 0.05) between the two groups on the preoperative day, they significantly differed (p < 0.05) between the two groups on the postoperative days. Further, the overall microbial distribution based on both unweighted and weighted principal coordinate analysis plots significantly differed (p < 0.05) between the two groups on both the preoperative and postoperative days. Orthogonal polynomial contrasts indicated that major genera and microbial diversity in the cannulation group decreased following surgery but returned to their initial states by postoperative day 28. In conclusion, this study demonstrates that rumen cannulation surgery affects some major taxa and microbial diversity, suggesting that the rumen cannulation method can alter the composition of rumen microbiota in Hanwoo steers.

Temporal changes in ewe vaginal microbiota throughout gestation.

Introduction: Numerous factors are known to influence reproductive efficiency in ewes, but few studies have investigated the potential role of vaginal microbiota in sheep reproductive success. The objective of this study was to thoroughly characterize the ewe vaginal microbiota throughout the course of pregnancy. Methods: Vaginal samples were collected from 31 pregnant Hampshire and Hampshire X Suffolk crossbred ewes on a weekly basis from pre-breeding to pregnancy testing and then biweekly until just after lambing. To characterize the vaginal microbial communities, DNA was extracted and 16S rRNA gene Illumina MiSeq amplicon sequencing was performed. Results and Discussion: Alpha diversity metrics indicated an increase in species richness, evenness, and overall diversity throughout gestation. Distinct shifts in the bacterial communities were observed during gestation and were segregated into three periods: early gestation, a transitional period and mid/late gestation. During early gestation, Actinobacillus, Histophilus, and unclassified Leptotrichiaceae were found in greater relative abundance. During the transitional period, a population shift occurred characterized by increasing relative abundance of Streptococcus and Staphylococcus. During mid/late gestation, Staphylococcus, Streptococcus, and Ureaplasma had the greatest relative abundance. These shifts in the microbial population throughout the ewe's gestation are likely related to hormonal changes triggered by the growing conceptus, specifically increasing blood concentration of progesterone. The transitional period shift in vaginal microbial communities potentially aligns with the placental take-over of progesterone production from the corpus luteum at approximately day 50 after conception (gestational week 7). Understanding the observed variability of the vaginal microbiota throughout pregnancy will allow for future comparison of ewes that did not become pregnant or had abnormal pregnancies, which could lead to the discovery of potential bacterial biomarkers for pregnancy outcome; this understanding could also lead to development of probiotics to improve sheep reproductive success.

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants.

BACKGROUND: Microorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. RESULTS: Here, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter. CONCLUSIONS: Our study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs. Video Abstract.

Blind spots of universal primers and specific FISH probes for functional microbe and community characterization in EBPR systems.

Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon sequencing are commonly used for microbial ecological analyses in biological enhanced phosphorus removal (EBPR) systems, the successful application of which was governed by the oligonucleotides used. We performed a systemic evaluation of commonly used probes/primers for known polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). Most FISH probes showed blind spots and covered nontarget bacterial groups. Ca. Competibacter probes showed promising coverage and specificity. Those for Ca. Accumulibacter are desirable in coverage but targeted out-group bacteria, including Ca. Competibacter, Thauera, Dechlorosoma, and some polyphosphate-accumulating Cyanobacteria. Defluviicoccus probes are good in specificity but poor in coverage. Probes targeting Tetrasphaera or Dechloromonas showed low coverage and specificity. Specifically, DEMEF455, Bet135, and Dech453 for Dechloromonas covered Ca. Accumulibacter. Special attentions are needed when using these probes to resolve the PAO/GAO phenotype of Dechloromonas. Most species-specific probes for Ca. Accumulibacter, Ca. Lutibacillus, Ca. Phosphoribacter, and Tetrasphaera are highly specific. Overall, 1.4% Ca. Accumulibacter, 9.6% Ca. Competibacter, 43.3% Defluviicoccus, and 54.0% Dechloromonas in the MiDAS database were not covered by existing FISH probes. Different 16S rRNA amplicon primer sets showed distinct coverage of known PAOs and GAOs. None of them covered all members. Overall, 520F-802R and 515F-926R showed the most balanced coverage. All primers showed extremely low coverage of Microlunatus (<36.0%), implying their probably overlooked roles in EBPR systems. A clear understanding of the strength and weaknesses of each probe and primer set is a premise for rational evaluation and interpretation of obtained community results.

Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats.

Different species of freshwater turtles exhibit primary behaviours ranging from aerial basking to benthic bottom-walking, cycle between wet and dry conditions at different time intervals, and undertake short-distance overland movements between aquatic habitats. These behaviours in turn may impact the accumulation of microbes on external shell surfaces of turtles and provide novel niches for differentiation of microbial communities. We assessed microbial diversity using 16S and 18S rRNA metabarcoding on carapace surfaces of six species of freshwater turtles residing in three adjacent and seasonally interconnected wetland habitats in southeast Oklahoma (United States). Communities were highly diverse, with nearly 4200 prokaryotic and 500 micro-eukaryotic amplicon sequence variants recovered, and included taxa previously reported as common or differentially abundant on turtle shells. The 16S rRNA alpha diversity tended to be highest for two species of benthic turtles, while 18S rRNA alpha diversity was highest for two basking and one shallow-water benthic species. Beta diversity of communities was more strongly differentiated by turtle species than by collection site, and ordination patterns were largely reflective of turtle species' primary habits (i.e. benthic, basking, or benthic-basking). Our data support that freshwater turtles could play a role in microbial ecology and evolution in freshwater habitats and warrant additional exploration including in areas with high native turtle diversity and inter-habitat turtle movements.

Addition of (bio)surfactants in the biofiltration of hydrophobic volatile organic compounds in air.

The removal of volatile organic compounds (VOCs) in air is of utmost importance to safeguard both environmental quality and human well-being. However, the low aqueous solubility of hydrophobic VOCs results in poor removal in waste gas biofilters (BFs). In this study, we evaluated the addition of (bio)surfactants in three BFs (BF1 and BF2 mixture of compost and wood chips (C + WC), and BF3 filled with expanded perlite) to enhance the removal of cyclohexane and hexane from a polluted gas stream. Experiments were carried out to select two (bio)surfactants (i.e., Tween 80 and saponin) out of five (sodium dodecyl sulfate (SDS), Tween 80, surfactin, rhamnolipid and saponin) from a physical-chemical (i.e., decreasing VOC gas-liquid partitioning) and biological (i.e., the ability of the microbial consortium to grow on the (bio)surfactants) point of view. The results show that adding Tween 80 at 1 critical micelle concentration (CMC) had a slight positive effect on the removal of both VOCs, in BF1 (e.g., 7.0 ± 0.6 g cyclohexane m-3 h-1, 85 ± 2% at 163 s; compared to 6.7 ± 0.4 g cyclohexane m-3 h-1, 76 ± 2% at 163 s and 0 CMC) and BF2 (e.g., 4.3 ± 0.4 g hexane m-3 h-1, 27 ± 2% at 82 s; compared to 3.1 ± 0.7 g hexane m-3 h-1, 16 ± 4% at 82 s and 0 CMC), but a negative effect in BF3 at either 1, 3 and 9 CMC (e.g., 2.4 ± 0.4 g hexane m-3 h-1, 30 ± 4% at 163 s and 1 CMC; compared to 4.6 ± 1.0 g hexane m-3 h-1, 43 ± 8% at 163 s and 0 CMC). In contrast, the performance of all BFs improved with the addition of saponin, particularly at 3 CMC. Notably, in BF3, the elimination capacity (EC) and removal efficiency (RE) doubled for both VOCs (i.e., 9.1 ± 0.6 g cyclohexane m-3 h-1, 49 ± 3%; 4.3 ± 0.3 g hexane m-3 h-1, 25 ± 3%) compared to no biosurfactant addition (i.e., 4.5 ± 0.4 g cyclohexane m-3 h-1, 23 ± 3%; hexane 2.2 ± 0.5 g m-3 h-1, 10 ± 2%) at 82 s. Moreover, the addition of the (bio)surfactants led to a shift in the microbial consortia, with a different response in BF1-BF2 compared to BF3. This study evaluates for the first time the use of saponin in BFs, it demonstrates that cyclohexane and hexane RE can be improved by (bio)surfactant addition, and it provides recommendations for future studies in this field.

Distinct microbial communities degrade cellulose diacetate bioplastics in the coastal ocean.

IMPORTANCE: Cellulose diacetate (CDA) is a promising alternative to conventional plastics due to its versatility in manufacturing and low environmental persistence. Previously, our group demonstrated that CDA is susceptible to biodegradation in the ocean on timescales of months. In this study, we report the composition of microorganisms driving CDA degradation in the coastal ocean. We found that the coastal ocean harbors distinct bacterial taxa implicated in CDA degradation and these taxa have not been previously identified in prior CDA degradation studies, indicating an unexplored diversity of CDA-degrading bacteria in the ocean. Moreover, the shape of the plastic article (e.g., a fabric, film, or foam) and plasticizer in the plastic matrix selected for different microbial communities. Our findings pave the way for future studies to identify the specific species and enzymes that drive CDA degradation in the marine environment, ultimately yielding a more predictive understanding of CDA biodegradation across space and time.

Tracking the contamination sources of microbial population and characterizing Listeria monocytogenes in a chicken slaughterhouse by using culture-dependent and -independent methods.

Listeria monocytogenes is the etiologic agent of listeriosis, a foodborne disease that poses a threat to public health globally. Chicken meat exhibits heightened susceptibility to L. monocytogenes contamination during butchery. The persistence of this pathogen in the slaughterhouse environment enables recurring contamination of meat products. This study aimed at identifying the sources and transmission routes of L. monocytogenes contamination within an abattoir where it was consistently detected for three consecutive years (2019-2021). Furthermore, the environmental factors aiding contamination along chicken processing lines were determined by surveying the microbiome within the facility. Samples collected in 2019 to 2021 were subjected to culture-dependent analysis to assess the prevalence, serotypes, and multi-locus sequence typing (MLST) of L. monocytogenes. Additionally, the specimens collected in 2021 underwent culture-independent analysis via real-time quantitative polymerase chain reaction (qPCR) and 16S rRNA gene amplicon sequencing to identify the contamination sources and characterize the entire microbial community within the slaughterhouse. L. monocytogenes was isolated only from the clean zone, where the final slaughtering stage occurs. Most strains isolated from the final carcasses showed the same genetic cluster as the isolate in the chilling water and were assigned to MLST profile ST3. Culture-independent qPCR confirmed L. monocytogenes contamination in all samples, excluding post-scalding carcasses, prewashed post-evisceration carcasses, and the bleeding areas. Consequently, qPCR enabled more comprehensive identification of L. monocytogenes contamination points than culture-dependent approaches. Moreover, 16S rRNA gene amplicon sequencing demonstrated that psychro-tolerant and spoilage-related bacteria with L. monocytogenes-like attributes exhibited enhanced viability in the clean zone and immersion-chilling water. Metagenomics-based source tracking analysis further revealed that the shackles and chilling waters represent predominant sources of cross-contamination between different slaughterhouse zones, whereas the grading and packaging workstations and chilling water in the clean zone were deemed crucial sources affecting final carcass contamination. Collectively, these findings demonstrate through culture-dependent and -independent methods that L. monocytogenes spreads along the slaughter line, contaminating the slaughterhouse. Moreover, by investigating changes in microbial community and bacterial flow along the slaughter line within the facility, the sources influencing carcass contamination can be effectively traced.