Competition for nutritional resources masks the true frequency of bacterial mutants

Background It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low. Results We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10−6/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results. Conclusion This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable “tragedy of the commons” case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar “tragedy of the commons-like” cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.

A bacterial tragedy of the commons that masks the actual frequency of mutants

The frequency of mutants in a population is central to the understanding of evolution. Mutant frequency is usually assessed by plating a bacterial culture on selective medium in which only specific rare mutants can grow, assuming that all mutant cells present on the plate are able to form colonies. Here we show an exception to this rule. Wild-type Escherichia coli cells are unable to grow with glycerol-2-phosphate (G2P) as a carbon source. In contrast, PHO-constitutive mutants can hydrolyse G2P to glycerol and form colonies on plates having G2P as their sole carbon source. However, the frequency of PHO-constitutive colonies on the selective plate is exceptionally low. Here we show that such mutations occur at a relatively high rate, but the growth of the existing mutants is inhibited due to a competition with the surrounding wild-type cells for the limited amounts of glycerol produced by the mutants. This scenario in which neither the wild-type nor the majority of the mutants are able to grow constitutes an unavoidable case of the ‘tragedy of the commons’. Evidence shows that the few mutants that do form colonies derive from micro-clusters of mutants on the selective plate. In addition, a mathematical model describes the fate of the wild-type and mutant populations on the selective plate.

The influence of agricultural treatment type on the microbial properties of sod-podzolic soil

In this study we examined the effects of conventional agricultural treatment with plowing and no-till treatment on the physical, chemical and microbiological properties of agro-transformed sod-podzolic loamy soil. Soil was sampled in eightfold spatial replication from the arable layers (0–10, 10– 20, 20–30 cm) of field No. 2 of the long-term field experiment of the Center for Precision Agriculture of the Russian State Agrarian University in June, 2018. The crop type on the field No. 2 was vetch and oat mix. Moisture content, water holding capacity, pH, percentage of carbon and nitrogen were determined. The NDVI vegetation index was measured using GreenSeeker HandHeld and used to estimate the plant development intensity. Microbiological properties were assessed by selective plate counts. The abundance and activity were estimated for the next ecological and trophic groups of microorganisms: heterotrophic ammonifiers, aerobic and anaerobic nitrogen-fixing agents, denitrifiers, oligotrophs, cellulolytics. The vegetation index NDVI was higher for plants growing on the plowed part of the field. The differences in microbiological properties when comparing soil samples under no-till and under plowing were insignificant (by t-test for the independent groups comparison). In no-till samples a greater number of micromycetes, including cellulolytic and phytopathogenic, was observed compared to conventional treatment. Profile distributions of bacterial and fungal gene abundances were similar for both treatments according to the paired comparison of samples from different layers. The similarity in microbiological properties was found in the condition of a higher moisture content of the arable layer of the soil and a higher percentage of nitrogen were revealed in the soil under no-till compared with the soil treated by plowing.

Comparative quantification of Campylobacter jejuni from environmental samples using traditional and molecular biological techniques

Campylobacter jejuni is one of the most common causes of gastroenteritis in the world. Given the potential risks to human, animal, and environmental health, the development and optimization of methods to quantify this important pathogen in environmental samples is essential. Two of the most commonly used methods for quantifying C. jejuni are selective plate counting and quantitative real-time PCR (qPCR). Unfortunately, little comparative research has been performed to evaluate the accuracy of these methods for quantification of C. jejuni in aqueous and solid matricies. In this study, the limit of detection and the level of resolution obtained using these 2 methods was evaluated for C. jejuni and compared with that of the common indicator organism Escherichia coli . The use of selective plate count media for quantification of C. jejuni resulted in a 0.7–1.2 log underestimation of cell concentrations, compared with qPCR in both water and column leachate samples, whereas E. coli concentrations were found to be similar with either technique. For C. jejuni, only the qPCR assay accurately measured 2-fold changes in cell concentrations in water samples, whereas concentrations of E. coli were accurately measured regardless of method. Based on these data, qPCR assays were found to be more accurate than selective plate counts for quantification of C. jejuni from environmental samples.

Survival and Recovery of Viable but Nonculturable Listeria monocytogenes Cells in a Nutritionally Depleted Medium

Survival of a desiccated five-strain Listeria monocytogenes mixture during storage in sand at 4°C for 2 months was determined using the acridine orange direct count method with novobiocin and plate counts. Samples of inoculated sand were taken every 2 weeks, incubated at 37°C for 6 h, stained with acridine orange, and then examined with a fluorescence microscope. Elongated viable but nonculturable cells were most frequently observed during weeks 2 and 4. At weeks 6 and 8, most of the cells either remained viable or were dead. In each microscopic field, only one or two viable but nonculturable cells were observed among hundreds of other viable culturable cells, indicating that L. monocytogenes does not generally become viable but nonculturable. Therefore, viable but nonculturable cells are not a concern when plating environmental samples or desiccated L. monocytogenes cells on nonselective media. Tryptic soy agar with 0.6% (wt/vol) yeast extract (TSAYE) and Columbia agar were used as nonselective plate count media. Modified Oxford agar and TSAYE + 5% (wt/vol) sodium chloride were used as the selective plate count media. The effects of aerobic or anaerobic incubation and media supplementation with0.1% or 1% (wt/vol) sodium pyruvate were tested to optimize recovery of desiccated cells. Nonselective media showed better recovery when TSAYE and Columbia agar contained 0.1% (wt/vol) pyruvate and were incubated aerobically. These two culture methods were equally effective (P > 0.05) for recovering desiccated L. monocytogenes cells.

Evaluation of VIDAS® Immuno-Concentration Salmonella (ICS) Plus Selective Plate Method (Hektoen Enteric, Bismuth Sulfite, Xylose Lysine Desoxycholate) for Detection of Salmonella in Selected Foods (Method Modification 2001.08): Collaborative Study

A new method for detection of Salmonella in foods in 48 h has been granted AOAC First Action approval in selected foods (Official Method 2001.08) using both the VIDAS Immuno-Concentration Salmonella (ICS) method and a combination of 3 selective plates: Hektoen enteric (HE), bismuth sulfite (BS), and xylose lysine desoxycholate (XLD).

Salmonella in Selected Foods by VIDAS® Immuno-Concentration Salmonella Plus Selective Plate Method (Hektoen Enteric, Xylose Lysine Desoxycholate, Bismuth Sulfite): Collaborative Study

The VIDAS Immuno-concentration Salmonella (ICS) plus selective plate method (Hektoen enteric, xylose lysine desoxycholate, bismuth sulfite) method for the detection of Salmonella was compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method in a collaborative study. Thirty-two laboratories participated in the evaluation. Each laboratory tested one or more of the 6 test products: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. The 2 methods were in agreement for 1297 of the 1455 samples. Of the 158 samples not in agreement, 82 were VIDAS ICS plus selective plate-positive and BAM/AOAC-negative, and 76 were VIDAS ICS plus selective plate-negative and BAM/AOAC-positive.