Population heterogeneity and dynamics in starter culture and lag phase adaptation of the spoilage yeast Zygosaccharomyces bailii to weak acid preservatives

The food spoilage yeast Zygosaccharomyces bailii shows great resistance to weak-acid preservatives, including sorbic acid (2, 4-hexadienoic acid). That extreme resistance was shown to be due to population heterogeneity, with a small sub-population of cells resistant to a variety of weak acids, probably caused by a lower internal pH reducing the uptake of all weak acids. In the present paper, it was found that resistant cells were extremely rare in exponential cultures, but increased by up to 8000-fold in stationary phase. Inoculation of media containing sorbic acid with a population of Z. bailii cells gave rise to what appeared to be a prolonged lag phase, suggesting adaptation to the conditions before the cells entered the period of exponential growth. However, the apparent lag phase caused by sorbic acid was largely due to the time required for the resistant sub-population to grow to detectable levels. The slow growth rate of the sub-population was identical to that of the final total population. The non-resistant bulk population remained viable for 3. days but had lost viability by 6. days and, during that time, there was no indication of any development of resistance in the bulk population. The sub-population growing in sorbic acid showed very high population diversity in colony size and internal pH. After removal of sorbic acid, the population rapidly reverted back to the normal, largely non-resistant, population distribution. The data presented suggest that a reevaluation of the lag phase in microbial batch culture is required, at least for the resistance of Z. bailii to sorbic acid. Furthermore, the significance of phenotypic diversity and heterogeneity in microbial populations is discussed more broadly with potential relevance to bacterial "persisters", natural selection and evolution. © 2014

Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures.

Based on two staining protocols, DiOC6(3)/ propidium iodide (PI) and RedoxSensor Green (an indicator of bacterial reductase activity)/PI, multiparameter flow cytometry and cell sorting has identified at least four distinguishable physiological states during batch cultures of Bacillus cereus. Furthermore, dependent on the position in the growth curve, single cells gave rise to varying numbers of colonies when sorted individually onto nutrient agar plates. These growing colonies derived from a single cell had widely different lag phases, inferred fromdifferences in colony size.This further highlights the complex population dynamics of bacterial monocultures and further demonstrates that individual bacterial cells in a culture respond in markedly dissimilar ways to the environment, resulting in a physiologically heterogenous and dynamic population

A bacteriophage detection tool for viability assessment of Salmonella cells

Available online 7 September 2013Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods

Assessment of physiological conditions in E. coli fermentations by epifluorescent microscopy and image analysis

The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes.Fundação para a Ciência e a Tecnologia (FCT) - POCI/BIO/60139/200

Freeing Pseudomonas putida KT2440 of its proviral load strengthens endurance to environmental stresses

2.6% of the genome of the soil bacterium Pseudomonas putida KT2440 encodes phage-related functions, but the burden of such opportunistic DNA on the host physiology is unknown. Each of the four apparently complete prophages borne by this strain was tested for stability, spontaneous excision and ability to cause lysis under various stressing conditions. While prophages P3 (PP2266-PP2297) and P4 (PP1532-1584) were discharged from the genome at a detectable rate, their induction failed otherwise to yield infective viruses. Isogenic P. putida KT2440 derivatives bearing single and multiple deletions of each of the prophages were then subjected to thorough phenotypic analyses, which generally associated the loss of proviral DNA with an increase of physiological vigour. The most conspicuous benefit acquired by prophage-less cells was a remarkable improvement in tolerance to UV light and other insults to DNA. This was not accompanied, however, with an upgrade of recA-mediated homologous recombination. The range of tolerance to DNA damage gained by the prophage-free strain was equivalent to the UV resistance endowed by the TOL plasmid pWW0 to the wild-type bacterium. While the P. putida's prophages are therefore genuinely parasitic, their detrimental effects can be offset by acquisition of compensatory traits through horizontal gene transfer.This study was supported by the BIO Program of the Spanish Ministry of Science and Innovation, the ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAM. The work in MK Laboratory is supported by Estonian Science Foundation, grant number 9114 to MK, by Estonian Ministry of Research Targeted Financing Project SF0180031s08.Peer reviewe

Use of flow cytometry and total viable count to determine the effects of orange juice composition on the physiology of Escherichia coli

Orange juice (OJ) contains numerous compounds some of which are known to play key roles in growth and survival of bacteria. This study aimed to investigate the effects of natural or processing‐induced variations in OJ composition on the physiology of Escherichia coli. OJ and model OJ (MOJ) samples containing various sugars, organic acids, amino acids, or ascorbic acid were inoculated with E. coli K‐12 MG1655 in different growth phases. The culturability, viability, and physiology of the cells were investigated during storage using plate counting and flow cytometry. Generally, stationary‐phase cells displayed the greatest survival in both MOJ and OJ. Increase in incubation temperature from 4 to 22.5ºC caused a significant decrease in both healthy and culturable cell populations. Supplementation of MOJ with ascorbic acid and amino acids increased both the viability and culturability of the cells. Similar trends were observed in amino acid‐supplemented OJ, albeit at a slower rate. In contrast, variations in sugar or organic acid composition had negligible effects on the physiological status of the cells. In summary, natural variation in ascorbic acid or amino acid concentrations could potentially have an adverse effect on the microbiological safety of orange juice

Flow cytometry for microbial sensing in environmental sustainability applications: current status and future prospects

Practical and accurate microbial assessment of environmental systems is predicated on the detection and quantification of various microbial parameters in complex matrices. Traditional growth-based assays, considered to be both slow and biased, are increasingly being replaced by optical detection methods such as flow cytometry. Flow cytometry (FCM) offers high-speed multi-parametric data acquisition, compatibility with current molecular-based microbial detection technologies, and is a proven technology platform. The unique technical properties of flow cytometry have allowed the discrimination of bacteria based on nucleic acid staining, microbial identification based on genomic and immunologic characteristics, and determination of cell viability. For this technology to achieve the ultimate goal of monitoring the microbial ecology of distributed systems, it will be necessary to develop a fully functional, low cost, and networkable microsystem platform capable of rapid detection of multiple species of microorganisms simultaneously under realistic environmental conditions. One such microsystem, miniaturized and integrated in accordance with recent advances in micro-electro-mechanical systems technology, is named the Micro Integrated Flow Cytometer. This manuscript is a minireview of the current status and future prospects for environmental application of flow cytometry in general, and micro-flow cytometry in particular.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75610/1/j.femsec.2004.01.014.pd