Microbiological indicators of water quality in submerged karst caves of Wakulla Springs

22 slides in Powerpoint presentation

When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining

Flow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered ‘dead’. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant

Disruption of cells in biosolids affects E. coli dynamics in storage

Achieving microbial compliance during biosolids storage can be complicated by the unpredictable increase of Escherichia coli. Thermal treatment during anaerobic digestion (AD) and the effects of dewatering may be a significant factor contributing to indicator survival. Shear forces present during dewatering may promote cell damage, releasing nutrient for E. coli growth. The effect of cell damage on E. coli survival was assessed in laboratory-scale thermal and physical disruption experiments. E. coli growth curves for disrupted treatments were compared with control conditions and quantified using flow cytometry and membrane filtration techniques. A significant difference (p < 0.05) in the level of damaged cells between control and disrupted conditions was observed. For thermal and physical disruption treatments, the peak of E. coli concentration increased significantly by 1.8 Log and 2.4 Log (CFU (colony forming units) g−1 DS), respectively, compared with control treatments. Research findings contribute to the understanding of bacterial growth and death dynamics in biosolid

Biodiversitätsmonitoring im Südtiroler Kräuteranbau = Biodiversity surveys in medicinal and aromatic plant fields in South Tyrol

Medicinal and aromatic plants in mountain regions such as South Tyrol are cultivated on small-scale farms, which are characterized by a high diversity of cultivated crop species grown on a relatively small area. This small-scale cultivation of medicinal and aromatic plants suggests that MAP fields are of high ecological value. However, research on this topic is generally lacking. In this study flower-visiting arthropods were recorded with pan traps in three herb fields during three survey events conducted in 2021. Our results indicate that medicinal and aromatic plant fields are valuable habitats for several taxa. In total 12.570 individuals were collected. Wild bees were particularly species-rich, accounting for 10 % of the regional wild bee species pool. Next to beneficial arthropods, potential pests, such as aphids were also highly abundant. However, natural enemies possibly counteracting pests were also numerous. Overall, we conclude that medicinal and aromatic plant cultivation may act as resource-rich oases for several arthropod groups, thereby promoting biodiversity also on a broader scale.Der Anbau von Arznei- und Gewürzpflanzen zeichnet sich in der Regel durch vielfältige Anbaukulturen auf relativ kleinen Flächen aus. Dies gilt insbesondere für Südtirol, wo diese Kulturen hauptsächlich von kleinen Betrieben im Berggebiet angebaut werden. Dieser kleinflächige Anbau von Arznei- und Gewürzpflanzen lässt vermuten, dass die Betriebe einen hohen ökologischen Wert haben. Es gibt wenige Studien zur Erfassung der Biodiversität im Anbau von Arznei- und Gewürzpflanzen. Daher wurden in dieser Arbeit Kräuteranbau-Betriebe als Lebensraum für blütenbesuchende Arthropoden untersucht. An drei Untersuchungsstandorten wurden im Jahr 2021 jeweils an drei Terminen Farbschalen zur Sammlung von Arthropoden verwendet. Kräuteranbau-Betriebe stellten sich als ein wertvoller Lebensraum für verschiedene Arthropoden heraus. Insgesamt wurden 12.570 Individuen mit den Farbschalen gesammelt. Insbesondere Wildbienen waren mit 10 % des regionalen Artenpools sehr artenreich. Auch potenzielle Schädlinge, wie zum Beispiel Blattläuse, waren sehr häufig anzutreffen, wobei natürliche Feinde, wie zum Beispiel Parasitoide, ebenfalls zahlreich vertreten waren. Insgesamt können Kräuteranbaubetriebe als strukturreiche Oasen für Arthropoden fungieren und sich somit auf einer breiteren Skala positiv auf die Biodiversität auswirken

The absence or presence of a lytic coliphage affects the response of Escherichia coli to heat, chlorine, or UV exposure

Disinfection aims at maximal inactivation of target organisms and the sustainable suppression of their regrowth. Whereas many disinfection efforts achieve efficient inactivation when the effect is measured directly after treatment, there are questions about the sustainability of this effect. One aspect is that the treated bacteria might recover and regain the ability to grow. In an environmental context, another question is how amenable surviving bacteria are to predation by omnipresent bacteriophages. Provisional data suggested that bacteria when subjected to sublethal heat stress might develop a phage-resistant phenotype. The result made us wonder about the susceptibility to phage-mediated lysis for bacteria exposed to a gradient of chlorine and UV-LED disinfection strengths. Whereas bacteria exposed to low sublethal chlorine doses still underwent phage-mediated lysis, the critical chlorine Ct of 0.5 mg min/L eliminated this susceptibility and induced phage resistance in the cells that survived treatment. In the case of UV, even the smallest tested dose of 2.8 mJ/cm2 abolished phage lysis leading to direct regrowth. Results suggest that bacteria surviving disinfection might have higher environmental survival chances directly after treatment compared to non-treated cells. A reason could possibly lie in their compromised metabolism that is essential for phage replication

The absence or presence of a lytic coliphage affects the response of Escherichia coli to heat, chlorine, or UV exposure

Disinfection aims at maximal inactivation of target organisms and the sustainable suppression of their regrowth. Whereas many disinfection efforts achieve efficient inactivation when the effect is measured directly after treatment, there are questions about the sustainability of this effect. One aspect is that the treated bacteria might recover and regain the ability to grow. In an environmental context, another question is how amenable surviving bacteria are to predation by omnipresent bacteriophages. Provisional data suggested that bacteria when subjected to sublethal heat stress might develop a phage-resistant phenotype. The result made us wonder about the susceptibility to phage-mediated lysis for bacteria exposed to a gradient of chlorine and UV-LED disinfection strengths. Whereas bacteria exposed to low sublethal chlorine doses still underwent phage-mediated lysis, the critical chlorine Ct of 0.5 mg min/L eliminated this susceptibility and induced phage resistance in the cells that survived treatment. In the case of UV, even the smallest tested dose of 2.8 mJ/cm2 abolished phage lysis leading to direct regrowth. Results suggest that bacteria surviving disinfection might have higher environmental survival chances directly after treatment compared to non-treated cells. A reason could possibly lie in their compromised metabolism that is essential for phage replication

Effect of temperature on bacteriophage-mediated lysis efficiency with a special emphasis on bacterial temperature history

Despite the great potential of phages as biocontrol agents, there is much uncertainty about the environmental factors influencing lysis efficiency. In this study we investigated the effect of temperature using three distinct lytic E. coli phages that were isolated from a single environmental water sample. All three were identified as dsDNA phages belonging to the Myoviridae family. Whereas the optimal growth temperature of E. coli is well known to be 37 ˚C and exposure of phages (prior to mixing with bacteria) to temperatures between 4 and 37˚C did not affect their infectivity, plaque sizes and numbers greatly decreased with increasing incubation temperature (20˚C, 30˚C, 37˚C) of the phage-host mix. At 37˚C, no visible plaques were observed. Results suggest that temperature sensitivity of the phage-host interaction is distinct from the temperature susceptibility of the two players and corroborate previous reports that highest lysis rates are obtained at temperatures approximate with ambient conditions of the phage environment. Infectivity was however found not only to depend on the incubation temperature of the phage-host mix, but also on the bacterial temperature history. Moreover, exposure of bacteria to heat stress prior to phage challenge resulted in a phage-resistant phenotype raising the question whether bacterial pathogens shed from warm-blooded hosts might be less susceptible to phages adapted to environmental temperature conditions

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo

Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.Comment: 28 pages, 13 figure

Modelling the metabolic effects of protective clothing

Protective clothing is worn in many industrial and military situations. Although worn for protection from one or more hazards, protective clothing can add significantly to the metabolic (energy) cost of work. Suggestions put forward as to the mechanisms behind the observed increases include, the additional clothing weight of the protective garments, possible friction between the number of layers that must be worn and restriction of movement due to clothing bulk. However, despite much speculation, these areas have not received much investigation. Wearing protective clothing from a range of industries and with quite different characteristics for example weight, bulk and stiffness significantly increased metabolic rate when walking, stepping and completing an obstacle course activity. Increases in the metabolic rate of up to 20% above control conditions (lightweight tracksuit and trainers worn) were seen. A number of clothing properties were then investigated to try and understand the causes of these recorded metabolic rate increases. Clothing bulk was measured at 3 sites, upper arm, torso and thigh. The stiffness of the clothing was also calculated, using a method which measured the clothing drape of the sleeve, main body of the garment and trouser leg. A multiple regression carried out on the data showed body weight to be the best predictor of absolute metabolic increases across all work modes. For the % increase in metabolic rate total clothing weight was the best predictor. Torso bulk was negatively correlated with the increased metabolic rate for walking and stepping and the overall average, whereas leg bulk was a significant predictor of an increased stepping metabolic rate and leg stiffness a significant predictor for the obstacle course work mode