Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Intracellular bacterial pathogens in aquaculture: vaccines and virulence factors

Fish farming is the most common form of aquaculture. Its production contributes close to 50% of total global fish production, and is estimated to increase to 62% by year 2030. Piscirickettsiosis and francisellosis are two common diseases in Atlantic salmon and cod respectively. These diseases arise due to the infection of pathogenic bacteria Piscirickettsia salmonis (piscirickettsiosis) and Francisella noatunensis subspecies noatunensis (francisellosis). Due to the absence of vaccines with a sufficiently long-lasting effect, the current strategy for both protection and treatment of these common aquaculture diseases is through the use of antibiotics. Unnecessary use of antibiotics is a global problem, and is an important factor in the emergence of multi-resistant bacterial strains. In this thesis, the focus has been on examining the effect from a vaccine trial against piscirickettsiosis in Atlantic salmon, using membrane vesicles isolated from P. salmonis, as well as evaluating the presence of a type IV pili system associated with virulence in F.n.n. Cumulative mortality obtained from the vaccine trial, indicates that no protective effect were induced by immunization with MVs. The reason for this was that no difference in onset of death nor total cumulative mortality were seen between control and vaccine groups, under the conditions tested. However, a potentially positive effect was detected for the immunized groups, as gene expression of both Interleukin-12 and macrophage-expressed gene were statistically significantly increased at 4 weeks post challenge. Histological analysis comparing tissue from immunized fish before and after challenge with P. salmonis did not show any apparent granulomas, otherwise commonly seen in fish infected with this pathogen. However, several other indications of disease could be seen in both spleen and kidney of infected fish, and could be related to the infection of P. salmonis, due to their presence only post infection. No granulomas were detected in neither control nor immunized group, and no defined differences in disease progression were seen between the groups. The presence of a type IV pili system was evaluated in F.n.n. by utilizing the methods immunogold labeling, atomic force microscopy, and flow cytometry analysis. F.n.n. mutants containing His-tagged pili proteins were used as markers for presence of pili, by the use of anti-His antibodies. Mutants containing a disrupted type IV pili component PilD along with His-tagged pilins, as well as mutants with only His-tagged pilins were examined. Disruption of the PilD pili subcomponent were investigated, to assess any difference in expression of pili. No pili nor type IV pili-like appendages were discovered using any of these techniques, on any of the mutants. However, heavy gold labeling was seen on the surface of several F.n.n. Bacteria positively labeled with the anti-His antibody were sorted using a flow cytometry, but neither an increase in gold labeling nor the presence of pili were seen. Because heavy labeling was present mainly in damaged or lysed bacteria, it is uncertain if this labeling is due to attachment to leaked internal components or if the condition itself leads to non-specific binding of antibodies

Neutral natural deep eutectic solvents as anti-biofilm agents

Natural deep eutectic solvents (NADES) are a class of liquids with promising properties as components in pharmaceutical formulations, such as a low toxicity profile, biodegradability and versatility. Recently, their potential use as anti-biofilm agents has been proposed, due to their ability to solubilize and stabilize biological macromolecules. In the current work, the ability to break down biofilm matrix and the biofilm killing activity of three NADES of neutral pH were investigated against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9027 biofilms. The tested NADES were choline chloride:xylitol (ChX), choline chloride:glycerol (ChG) and betaine:sucrose (BS). Two of the NADES (ChX and ChG) significantly reduced the number of remaining viable cells of both bacterial species in pre-formed biofilm by 4–6 orders of magnitude, while the average biofilm biomass removal for all NADES was 27–67% (S. aureus) and 34–49% (P. aeruginosa). The tested NADES also inhibited biofilm formation of both bacterial species at concentrations at or below 0.5 x the minimal inhibitory concentration (MIC), possibly in part due to observed restrictions imposed by NADES on planktonic growth. These results demonstrate the potential value of neutral NADES as anti-biofilm agents in future antimicrobial preparations