Total Bacterial Cell Research Articles

You can bring plankton to fecal indicator organisms, but you cannot make the plankton graze: particle contribution to E. coli and MS2 inactivation in surface waters.

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 ("E. coli") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day-1 in the marine waters and -0.89 to -3.2 day-1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators.

PSV-12 Evaluating the effect of lyoprotectants in preserving community structure and bacterial viability in the lyophilized fecal community of pigs

Abstract Fecal microbiota transplantation (FMT) stands as a potential method for enhancing pig health. FMT has progressed from the basic delivery of fresh stool, to lyophilized preparations which is beneficial for sample storage, but can damage bacterial cells. This has necessitated the need for lyoprotectants to preserve bacterial viability. However, the ideal lyoprotectant for retaining community structure and viability remains unclear. Furthermore, DNA-based microbial profiling methods such as 16S rRNA and metagenomics sequencing do not distinguish live and dead bacteria, prompting the need for other methods of distinguishing live and dead members of the bacterial community after lyophilization. PMA-seq (propidium monoazide treatment followed by 16S rRNA gene amplicon sequencing) has been shown to differentiate between live and dead bacteria in microbial communities. To refine FMT in pigs, we sought to ascertain the impact of four lyoprotectants on bacterial viability and fecal microbiome structure. Fecal samples from six pigs were treated with mannitol, maltodextrin, trehalose, maltodextrin-trehalose mix (2.5% each) as lyoprotectants or left untreated. Lyophilized fecal samples underwent PMA treatment prior to 16S rRNA sequencing to capture the active community, and total and viable bacterial cells number were quantified. One-way ANOVA was used to analyze total and viable bacterial cells, and richness, while PERMANOVA was used to compare beta diversity. Results revealed no initial differences in total, viable bacteria, or community structure (P > 0.05). However, untreated, and mannitol-treated groups showed significantly reduced total and viable bacterial cells post-lyophilization (P < 0.01), unlike the other lyoprotectants (P > 0.05). Mannitol and the untreated group also displayed decreased richness and beta diversity shifts (P < 0.05). Overall, lyophilization with mannitol or no lyoprotectant notably impacted pig fecal microbiota community structure, while maltodextrin, trehalose, and maltodextrin-trehalose effectively preserved bacterial viability and structure.

Investigating drug-gut microbiota interactions: reductive and hydrolytic metabolism of oral glucocorticoids by in vitro artificial gut microbiota

Elucidation of the role of gut microbiota in the metabolism of orally administered drugs may improve therapeutic effectiveness and contribute to the development of personalized medicine. In this study, ten different artificial gut microbiota (AGM), obtained by culturing fecal samples in a continuous fermentation system, were challenged for their metabolizing capacity on a panel of six glucocorticoids selected from either prodrugs or drugs. Data from metabolic stability assays highlighted that, while the hydrolysis-mediated conversion of prodrugs to drugs represented only a minor metabolic pathway, significant differences in the stability of parent compounds and in their conversion rates to multiple reductive metabolites were obtained for the selected drugs. In the latter case, a taxonomic composition-dependent ability to convert parent drugs to metabolites was observed. Indeed, the artificial microbial communities dominated by the genus Bacteroides showed the maximal conversion of parent glucocorticoids to several metabolites. Furthermore, the effect of drugs on AGM was also evaluated through shallow shotgun sequencing and flow cytometry-based total bacterial cell count highlighting that these drugs can affect both the taxonomic composition and growth performances of the human gut microbiota.

Targeted remodeling of the human gut microbiome using Juemingzi (Senna seed extracts).

The genus Senna contains globally distributed plant species of which the leaves, roots, and seeds have multiple traditional medicinal and nutritional uses. Notable chemical compounds derived from Senna spp. include sennosides and emodin which have been tested for antimicrobial effects in addition to their known laxative functions. However, studies of the effects of the combined chemical components on intact human gut microbiome communities are lacking. This study evaluated the effects of Juemingzi (Senna sp.) extract on the human gut microbiome using SIFR® (Systemic Intestinal Fermentation Research) technology. After a 48-hour human fecal incubation, we measured total bacterial cell density and fermentation products including pH, gas production and concentrations of short chain fatty acids (SCFAs). The initial and post-incubation microbial community structure and functional potential were characterized using shotgun metagenomic sequencing. Juemingzi (Senna seed) extracts displayed strong, taxon-specific anti-microbial effects as indicated by significant reductions in cell density (40%) and intra-sample community diversity. Members of the Bacteroidota were nearly eliminated over the 48-hour incubation. While generally part of a healthy gut microbiome, specific species of Bacteroides can be pathogenic. The active persistence of the members of the Enterobacteriaceae and selected Actinomycetota despite the reduction in overall cell numbers was demonstrated by increased fermentative outputs including high concentrations of gas and acetate with correspondingly reduced pH. These large-scale shifts in microbial community structure indicate the need for further evaluation of dosages and potential administration with prebiotic or synbiotic supplements. Overall, the very specific effects of these extracts may offer the potential for targeted antimicrobial uses or as a tool in the targeted remodeling of the gut microbiome.

Influence of Season on Biodegradation Rates in Rivers.

Biodegradation plays a key role in the fate of chemicals in the environment. The variability of biodegradation in time can cause uncertainty in evaluating the environmental persistence and risk of chemicals. However, the seasonality of biodegradation in rivers has not yet been the subject of environmentally relevant testing and systematic investigation for large numbers of chemicals. In this work, we studied the biodegradation of 96 compounds during four seasons at four locations (up- and downstream of WWTPs located on two Swedish rivers). Significant seasonality (ANOVA, p < 0.05) of the first-order rate constant for primary biodegradation was observed for most compounds. Variations in pH and total bacterial cell count were not the major factors explaining the seasonality of biodegradation. Deviation from the classical Arrhenius-type behavior was observed for most of the studied compounds, which calls into question the application of this relationship to correct biodegradation rate constants for differences in environmental temperature. Similarities in magnitude and seasonality of biodegradation rate constants were observed for some groups of chemicals possessing the same functional groups. Moreover, reduced seasonality of biodegradation was observed downstream of WWTPs, while biodegradation rates of most compounds were not significantly different between up- and downstream.

Microbial communities in the fermentation of Meju, a Korean traditional soybean brick.

Meju is a traditional Korean soybean brick characterized by diverse microbial communities. The microbial communities in Meju were identified at the phylum and genus levels using high-throughput sequencing. During Meju fermentation, diverse factors such as total bacterial cell numbers, moisture content, salinity, pH, enzyme activities, and free amino acids were monitored. After 30days of fermentation, microbial adaptation and increased protease activity resulted in significant changes, including an increase in pH and alterations in free amino acid content by day 70. Bacterial community analysis revealed significant changes in Bacillus, Lactococcus, and Enterococcus levels as fermentation progressed. The decrease in pH during fermentation was influenced by lactic acid bacteria, which affected bacterial dynamics. At the end of fermentation, the fungal community was dominated by Monascus, Aspergillus, and Scopulariopsis, which affected the free amino acid levels. These results indicate that pH and moisture content may be significant factors in determining microbial communities.

Effects of probiotics on biogenic amines content in fermented milk during fermentation and storage

This study aimed to investigate the formation of biogenic amines (BAs) during fermentation and storage in fermented milk. The traditional culture was used as a control, and three different probiotics were added to the above culture as different experimental groups. Results revealed that the degree of protein hydrolysis in fermented milk increased during fermentation but decreased during storage, reaching a maximum value (14.8–17.3%) at the end of fermentation. The trends of total amino acid content and total BAs content were similar to those of the degree of hydrolysis. The total amino acid content reached a maximum value (2.83–3.22 mg/mL) after 24 h of post-ripening. Fermented milk had the highest tyramine content, but it was always below 10 mg/L. The total BAs content in fermented milk during fermentation and storage ranged from 9.04 to 15.2 mg/L and 6.37 to 14.0 mg/L, respectively. These levels consistently remained below the threshold considered harmful to humans. The pH reduction rate, the total bacterial cell number, the degree of hydrolysis, and the total amino acid content in fermented milk all affected the formation and content of BAs. This study provides a foundation for assessing the potential public health impact of BAs in fermented milk.

Metataxonomic analysis of milk microbiota in the bovine subclinical mastitis.

Subclinical mastitis is one of the most widespread diseases affecting dairy herds with detrimental effects on animal health, milk productivity, and quality. Despite its multifactorial nature, the presence of pathogenic bacteria is regarded one of the main drivers of subclinical mastitis, causing a disruption of the homeostasis of the bovine milk microbial community. However, bovine milk microbiota alterations associated with subclinical mastitis still represents a largely unexplored research area. Therefore, the species-level milk microbiota of a total of 75 milk samples, collected from both healthy and subclinical mastitis-affected cows from two different stables, was deeply profiled through an ITS, rather than a traditional, and less informative, 16S rRNA gene microbial profiling. Surprisingly, the present pilot study not only revealed that subclinical mastitis is characterized by a reduced biodiversity of the bovine milk microbiota, but also that this disease does not induce standard alterations of the milk microbial community across stables. In addition, a flow cytometry-based total bacterial cell enumeration highlighted that subclinical mastitis is accompanied by a significant increment in the number of milk microbial cells. Furthermore, the combination of the metagenomic and flow cytometry approaches allowed to identify different potential microbial marker strictly correlated with subclinical mastitis across stables.

High-Throughput Viability Testing of Microbial Communities in a Probiotic Product Using Flow Cytometry

There is growing scientific and commercial interest in multi-species probiotic products due to their potential benefits in maintaining gut health. Determining the viability of probiotic microorganisms in these products is essential to ensure that they confer maximal health benefits. The gold standard for enumerating probiotic viability is the plate count method. However, this may be inaccurate for enumerating mixed probiotic populations, with recognised limitations including difficulty measuring metabolically active yet unculturable, very slow growing microbes, microencapsulated, enteric coated microbes, or multi-strain formulations that require differing growth media. Here, we developed a flow-cytometry-based approach using SYTOXTM Green dye to assess the viability of probiotic microorganisms in a multi-species, fibre-containing probiotic product and compared this to the traditional plate count method. This method was suitable for enumerating both total bacterial cells and the viable cell fraction in the complete product mixture, and could also be used to assess how stressors, such as gastric digestion and exposure to bile acids, affect bacterial cell viability. Flow cytometry measurements routinely detected higher viable cell counts than plate counting. This work demonstrates that flow cytometry assays can be established as a suitable method for rapid enumeration of viable cells in complex, multi-species probiotics.

Anti-biofilm activity of a novel nanoemulsion containing Curcuma xanthorrhiza oil

ObjectivesWe aimed to solubilize Curcuma xanthorrhiza oil (CXO) using nanoemulsification and evaluate its inhibitory effects against biofilm formation. MethodsThe components of CXO were evaluated through high-performance liquid chromatography (HPLC) analysis. Healthy human saliva was inoculated onto hydroxyapatite discs to form microcosm biofilms for four days and treated six times with each antimicrobial agent: distilled water (DW), CXO emulsion (EM), CXO nanoemulsion (NE), and positive controls (Listerine and chlorhexidine). Biofilm fluorescence imaging was performed using quantitative light-induced fluorescence, and cell viability and dry-weight measurements were obtained. We compared the bacterial cell and extracellular polysaccharide (EPS) biovolume and thickness using confocal laser scanning microscopy (CLSM). ResultsHPLC analysis revealed that CXO was composed of approximately 47% xanthorrhizol. Compared with DW, NE exhibited significantly lower red fluorescence intensity and area (42% and 37%, p < 0.001 and p < 0.001, respectively), and reduced total and aciduric bacterial cell viability (7.3% and 3.9%, p < 0.001, p = 0.01, respectively). Furthermore, the bacterial cell and EPS biovolume and thickness in NE decreased by 40–80% compared to DW, similar to chlorhexidine. Conversely, EM showed a significant difference only in cell viability against total bacteria when compared with DW (p = 0.003), with EPS biovolume and thickness exhibiting higher values than DW. ConclusionsNanoemulsification successfully solubilized CXO and demonstrated superior anti-biofilm effects compared to the emulsion form. Clinical significanceThese findings suggest the potential use of NE as a novel antimicrobial agent for preventing oral diseases.

Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

Covariates of vaginal microbiota and pro-inflammatory cytokine levels in women of reproductive age.

This study aimed to assess the correlation between covariates of the vaginal microbiota and local levels of proinflammatory cytokines in women of reproductive age presenting four molecularly defined bacterial community-state types (CSTs). We enrolled 133 non-pregnant women who attended primary care health clinics for routine Pap-testing. Molecular profiling of vaginal microbiota was performed by V3-V4 16S rRNA sequencing. The covariates of vaginal microbiota included were: vaginal pH, total bacterial cell count, diversity (Shannon index), -richness and dominant taxa abundances. Levels of interleukin (IL)-1β, IL-6, IL-8, and tumour necrosis factor (TNF-α) were measured by enzyme-linked immunosorbent assays in supernatants of cervicovaginal fluids. Nonparametric Kruskal-Wallis test was used to compare microbiota covariates and cytokines among different CSTs. Spearman's tests were performed to assess correlations across the measured parameters. A total of 96 (72.2%) participants had CSTs dominated by Lactobacillus spp. (Lactobacillus crispatus CST I, n=38; Lactobacillus gasseri CST II, n=20; and Lactobacillus iners CST III, n=38). A total of 37 (27.8%) presented the Lactobacillus-depleted CST IV. Total bacterial count was higher in CST II (1.29E+05, 3.40E+04-6.69E+05) compared to other Lactobacillus-dominated CSTs (p=0.0003). The highest values of microbiota diversity (1.85; 0.23-2.68) and richness (27.0; 5.0-37.0) were observed in CST IV (P<0.0001). Lower levels of IL-1β were observed in CST I (5.4; 0.0-3,256) when compared to CST III (51.7; 0.0-2,616) and to CST IV (56.2; 0.0-3,407) (P=0.008). Levels of IL-6 were higher in CST II (4.13; 0-131.4) than in CST IV (0.0-58.27) (P=0.02). Correlation tests showed an overall distinct profile of CST II when compared to other Lactobacillusdominated CSTs, particularly regarding the correlation between total bacterial load and cytokines (r>0.39). In conclusion, this study provides evidence of a single pro-inflammatory signature of L. gasseri-dominated microbiota in response to bacterial load. Further studies evaluating a broader range of inflammation markers are warranted.

Association Between Bacterial Count and Polymorphonuclear Cells in Uterine Lavage of Postpartum Subclinical Endometritic Cows

This study was conducted to find out the total bacterial count and percentage of polymorphoneutrophils (PMN) cells in uterine lavage samples obtained at a weekly interval from postpartum dairy cows and also to find out the correlation between total bacterial count and PMN cells recovered from the uterus of postpartum dairy cows. Twenty-one postpartum dairy cows were divided into two groups viz. group A (without postpartum complication; n=16) and group B (retained placenta/postpartum metritis; n=5) calving. Cows belonging to the group B were administered antibiotics for 5 days after parturition. Samples for endometrial cytology and total bacterial count were collected aseptically by uterine lavage method at weekly intervals from day 21 to 42 postpartum. Data were statistically analysed by using one-way ANOVA, correlation using Statistical Analysis Software, SAS® 9.2 TS Level version 2M2 for Windows (USA). Based on endometrial cytology, the incidence of sub-clinical endometritis (SCE) recorded was 18.75% in group A and 100% in cows with postpartum complications (Group B). Total bacterial count in the group A animals differed significantly (P&lt;0.05) with the group-B animals. However, the difference between the total bacterial counts in group-A animals and diagnosed negative or positive for SCE did not differ significantly. A highly significant (P&lt;0.05 and P&lt;0.01) correlation was found between bacterial load and PMN cells obtained by uterine lavage technique.

Cobalt oxide nanoparticles as a new strategy for enhancing methane production from anaerobic digestion of noxious aquatic weeds

This study investigated the effect of cobalt oxide nanoparticles (Co3O4-NPs) supplementation on anaerobic microbial population changes and anaerobic digestion (AD) performance and production. Co3O4-NPs (3 mg/L) showed the maximum enhancement of biogas yield over the cow dung (CD) as control and the co-digestion process of CD with water hyacinth (WH) by 58.9 and 27.2 %, respectively. Furthermore, methane (CH4) yield was enhanced by 89.96 and 43.4 % over CD and co-digestion processes, respectively. Additionally, the microbiological assessment analysis using VIT® gene probe technology showed that Co3O4-NPs enhance the viability of total bacterial cells by 9 %. The techno-economic analysis reflects the revenue of this strategy on the highest net energy content of biogas, which was achieved with 3 mg/L Co3O4-NPs and was 428.05 kWh with a net profit of 67.66 USD/m3 of the substrate. Therefore, nanoparticle supplementation to the AD process can be considered a promising approach to enhance biogas and CH4.

Deciphering longitudinal optical-density measurements to guide clinical dosing regimen design: A model-based approach

BackgroundModel-based analysis of longitudinal optical density measurements from a bacterial suspension exposed to antibiotics has been proposed as a potentially efficient and effective method for extracting useful information to improve the individualized design of treatments for bacterial infections. To that end, the authors developed in previous work a mathematical modeling framework that can use such measurements for design of effective dosing regimens. ObjectivesHere we further explore ways to extract information from longitudinal optical density measurements to predict bactericidal efficacy of clinically relevant antibiotic exposures. MethodsLongitudinal optical density measurements were collected in an automated instrument where Acinetobacter baumannii, ATCC BAA747, was exposed to ceftazidime concentrations of 1, 4, 16, 64, and 256 mg/L and to ceftazidime/amikacin concentrations of 1/0.5, 4/2, 16/8, 64/32, and 256/128 (mg/L)/(mg/L) over 20 h. Calibrated conversion of measurements produced total (both live and dead) bacterial cell concentration data (CFU/mL equivalent) over time. Model-based data analysis predicted the bactericidal efficacy of ceftazidime and of ceftazidime/amikacin (at ratio 2:1) for periodic injection every 8 h and subsequent exponential decline with half-life of 2.5 h. Predictions were experimentally tested in an in vitro hollow-fiber infection model, using peak concentrations of 60 and 150 mg/L for injected ceftazidime and of 40/20 (mg/L)/(mg/L) for injected ceftazidime/amikacin. ResultsModel-based analysis predicted low (<62%) confidence in clinically relevant suppression of the bacterial population by periodic injections of ceftazidime alone, even at high peak concentrations. Conversely, analysis predicted high (>95%) confidence in bacterial suppression by periodic injections of ceftazidime/amikacin combinations at a wide range of peak concentrations ratioed at 2:1. Both predictions were experimentally confirmed in an in vitro hollow fiber infection model, where ceftazidime was periodically injected at peak concentrations 60 and 150 mg/L (with predicted suppression confidence 38% and 59%, respectively) and a combination of ceftazidime/amikacin was periodically injected at peak concentrations 40/20 (mg/L)/(mg/L) (with predicted suppression confidence 98%). ConclusionsThe paper highlights the potential of clinicians using the proposed mathematical framework to determine the utility of different antibiotics to suppress a patient-specific isolate. Additional studies will be needed to consolidate and expand the utility of the proposed method.

MALDI-TOF MS for pathogenic bacteria analysis

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been widely used for bacterial identification in clinical laboratories. However, the current technique needs bacterial culture to obtain purified single colonies for MS analysis. It is also limited in direct bacterial antimicrobial resistance (AMR) analysis. The two limitations restrict fast clinical diagnosis of bacterial infectious diseases and the choice of suitable antibiotic drugs in a timely manner. In the past years, we focus on bacterial identification and bacterial AMR analysis by MALDI-TOF MS, and developed a number of new methods with respect to sample pretreatment, mass spectrometry data acquisition and mass spectrometry data mining, with the aim of more accurate and more rapid bacterial identification as well as direct bacterial AMR analysis. Microfluidic chips and functional nanomaterials can be used to extract bacterial cells directly from the raw materials to shorten or even avoid the bacterial culture step. In some sample, the total bacterial cell amount is limited and hence it is necessary to further enhance the MS analysis sensitivity, and the single cell mass spectrometry techniques may play an important role in bacterial identification in the future. In view of bacterial AMR analysis, it is necessary to collect more molecular information of bacterial cells by mass spectrometry and to develop advanced data mining strategies based on machine learning and deep learning techniques. It is expected that the MALDI-TOF MS technique can further advance clinical treatment of bacterial infection diseases in combination with the development of sample pretreatment methods, MS techniques and data analysis algorithms.

Online evaluation of bacterial cells in sand filter effluents during full-scale treatment of drinking water

Ensuring the microbiological safety of drinking water is critical to protect public health. This study aimed to evaluate the reliability of real-time bacteriological counter coupled with an online dialysis membrane-based pre-treatment system for continuously monitoring bacterial cell counts in sand filter effluents of a full-scale drinking water treatment plant. The pre-treatment system, which included anion exchange resins (porous polymeric microbeads that trap ions for releasing other ions) for dialysate regeneration, successfully achieved the stable attenuation of background interfering substances (humic acids) during the 19-d test. The real-time bacteriological counter equipped with the pre-treatment system provided a continuous profile of bacterial cell counts in the sand filter effluent (0.2–2.5 × 104 counts/mL). The online analysis identified different timing of concentration peaks between particle and bacterial cell counts after backwashing. Bacterial community analysis revealed that Proteobacteria, Planctomycetes, and Cyanobacteria were the dominating phyla. Further, total bacterial cell counts determined by fluorescence microscopy and SYBR® Green I staining, a commonly accepted parameter, was found to be an indicator of online-monitored bacterial cell counts. The results indicated the potential of monitoring the bacterial cell counts in a sand filter process for providing an early warning of filter failures, which can allow plant operators to diagnose the overall system and provide countermeasures.

Specific detection and quantification of the marine flavobacterial genus Zobellia on macroalgae using novel qPCR and CARD-FISH assays

The flavobacterial genus Zobellia is considered as a model to study macroalgal polysaccharide degradation. The lack of data regarding its prevalence and abundance in coastal habitats constitutes a bottleneck to assess its ecological strategies. To overcome this issue, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) methods targeting the 16S rRNA gene were optimized to specifically detect and quantify Zobellia on the surface of diverse macroalgae. The newly designed qPCR primers and FISH probes targeted 98 and 100% of the Zobellia strains in silico and their specificity was confirmed using pure bacterial cultures. The dynamic range of the qPCR assay spanned 8 orders of magnitude from 10 to 108 16S rRNA gene copies and the detection limit was 0.01% relative abundance of Zobellia in environmental samples. Zobellia-16S rRNA gene copies were detected on all surveyed brown, green and red macroalgae, in proportion varying between 0.1 and 0.9% of the total bacterial copies. The absolute and relative abundance of Zobellia varied with tissue aging on the kelp Laminaria digitata. Zobellia cells were successfully visualized in Ulva lactuca and stranded Palmaria palmata surface biofilm using CARD-FISH, representing in the latter 105Zobellia cells·cm−2 and 0.43% of total bacterial cells. Overall, qPCR and CARD-FISH assays enabled robust detection, quantification and localization of Zobellia representatives in complex samples, underlining their ecological relevance as primary biomass degraders potentially cross-feeding other microorganisms.

In-situ fluorescence spectroscopy is a more rapid and resilient indicator of faecal contamination risk in drinking water than faecal indicator organisms

Faecal indicator organisms (FIOs) are limited in their ability to protect public health from the microbial contamination of drinking water because of their transience and time required to deliver a result. We evaluated alternative rapid, and potentially more resilient, approaches against a benchmark FIO of thermotolerant coliforms (TTCs) to characterise faecal contamination over 14 months at 40 groundwater sources in a Ugandan town. Rapid approaches included: in-situ tryptophan-like fluorescence (TLF), humic-like fluorescence (HLF), turbidity; sanitary inspections; and total bacterial cells by flow cytometry. TTCs varied widely in six sampling visits: a third of sources tested both positive and negative, 50% of sources had a range of at least 720 cfu/100 mL, and a two-day heavy rainfall event increased median TTCs five-fold. Using source medians, TLF was the best predictor in logistic regression models of TTCs ≥10 cfu/100 mL (AUC 0.88) and best correlated to TTC enumeration (ρs 0.81), with HLF performing similarly. Relationships between TLF or HLF and TTCs were stronger in the wet season than the dry season, when TLF and HLF were instead more associated with total bacterial cells. Source rank-order between sampling rounds was considerably more consistent, according to cross-correlations, using TLF or HLF (min ρs 0.81) than TTCs (min ρs 0.34). Furthermore, dry season TLF and HLF cross-correlated more strongly (ρs 0.68) than dry season TTCs (ρs 0.50) with wet season TTCs, when TTCs were elevated. In-situ TLF or HLF are more rapid and resilient indicators of faecal contamination risk than TTCs.

Potential of Bacillus cereus SN7 as a Single Cell Protein Source

Single-cell protein (SCP) is a protein source produced from single-cell organisms, one of which is bacteria. Bacillus cereus SN7 is a potential isolate from a group of heterotrophic bacteria that has been isolated from the mouth of the Siak River, Riau Province, Indonesia. This study aims to analyze the potential of B. cereus SN7 bacteria using different growth media. The method used is an experimental method using Bacillus cereus SN7 bacterial isolate with different protein sources (eggs and skim milk) at different concentrations (8%, 10%, and 12%). All of treatments used the same carbohydrate source, Sago with 3 replications in each treatment. Measurement of bacterial culture growth was carried out every 6 hours, 12 hours, 18 hours and 24 hours based on the total plate count (TPC) method and bacterial cell biomass. The result show that the most optimal growth potential for Bacillus cereus SN7 bacteria is sago media added with 12% egg white as a protein source. This data have the same results as growth in commercial culture media (control). The growth of Bacillus cereus SN7 isolates in each treatment medium had almost the same growth pattern at the beginning, which experienced an exponential phase at 6 to 12 hours of incubation time. Meanwhile, in milk sago media, the average growth was not very good because the exponential and stationary phases were shorter.