Ayse Gul Gozen

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

ABSTRACT Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with Escherichia coli ATCC 25922 as the model organism and the uidA gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.

High level multiple antibiotic resistance among fish surface associated bacterial populations in non-aquaculture freshwater environment.

Freshwater fish, Alburnus alburnus (bleak), were captured from Lake Mogan, situated in Ankara, during spring. The surface mucus of the fish was collected and associated bacteria were cultured and isolated. By sequencing PCR-amplified 16S RNA encoding genes, the isolates were identified as members of 12 different genera: Acinetobacter, Aeromonas, Bacillus, Brevundimonas, Gordonia, Kocuria, Microbacterium, Mycobacterium, Pseudomonas, Rhodococcus, and Staphylococcus, in addition to one strain that was unidentified. The mucus-dwelling bacterial isolates were tested for resistance against ampicillin, kanamycin, streptomycin and chloramphenicol. About 95% of the isolates were found to be resistant to ampicillin, 93% to chloramphenicol, and 88% to kanamycin and streptomycin. A Microbacterium oxydans and the unidentified environmental isolate were resistant to all four antibiotics tested at very high levels (>1600 μg/ml ampicillin and streptomycin; >1120 μg/ml kanamycin; >960 μg/ml chloramphenicol). Only a Kocuria sp. was sensitive to all four antibiotics at the lowest concentrations tested (3.10 μg/ml ampicillin and streptomycin; 2.15 μg/ml kanamycin; 1.85 μg/ml chloramphenicol). The rest of the isolates showed different resistance levels. Plasmid isolations were carried out to determine if the multiple antibiotic resistance could be attributed to the presence of plasmids. However, no plasmid was detected in any of the isolates. The resistance appeared to be mediated by chromosome-associated functions. This study indicated that multiple antibiotic resistance at moderate to high levels is common among the current phenotypes of the fish mucus-dwelling bacterial populations in this temperate, shallow lake which has not been subjected to any aquaculturing so far but under anthropogenic effect being in a recreational area.

Synthesis of 2-aziridinyl phosphonates by modified Gabriel—Cromwell reaction and their antibacterial activities

A set of new aziridinyl phosphonates (4a-g) were synthesized by using the Gabriel-Cromwell reaction and its modified version developed in this study and their structures confirmed by HRMS, IR, and NMR spectra. All the compounds were screened for their antibacterial activity. They all showed comparable moderate to good growth inhibitory activity in reference to ampicillin and streptomycin.

FTIR spectroscopy offers hints towards widespread molecular changes in cobalt-acclimated freshwater bacteria

High concentrations of heavy metals can be toxic for bacteria. However, after prolonged exposure, bacteria can become acclimated and begin to be able to grow in the presence of heavy metals. Acclimation can involve alterations of metabolism and molecular structures. Our aim was to examine these alterations in cobalt-acclimated bacteria via attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy on viable samples. Bacillus sp. and Pseudomonas sp. isolated from a temperate shallow lake and a well-established strain of E. coli were investigated. Our results revealed consistent, wide-spread changes in cell membrane and cell wall dynamics of Bacillus sp. and E. coli, including a decrease in peptidoglycan content of Bacillus sp. and increased lipid ordering of the membrane in both bacteria. Furthermore, a decrease in RNA and protein concentrations of Bacillus sp. was measured. All three bacteria studied showed a decrease in conformational freedom of proteins following cobalt acclimation. Interestingly, both Bacillus sp. and E. coli showed slight but significant alterations in their DNA conformations which might imply a methylation-mediated memory formation leading to epigenetic modulation for cobalt adaptation.

Impact of food concentration on diel vertical migration behaviour of Daphnia pulex under fish predation risk

Vertical migration of Daphnia represents the best-studied predator-avoidance behaviour known; yet the mechanisms underlying the choice to migrate require further investigation to understand the role of environmental context. To investigate the optimal habitat choice of Daphnia under fish predation pressure, first, we selected the individuals exhibiting strong migration behaviour. The animals collected from the hypolimnion during the daytime were significantly larger, being more conspicuous, and in turn performed stronger diel vertical migration (DVM) when exposed to fish cue. We called them strong migrants. Second, we provided the strong migrant D. pulex with food at high and intermediate (1 and 0.4 mg C l−1, respectively) levels, which were well above the incipient limiting level and of high quality. They traded the benefits of staying in the warm water layer and moved down to the cold water in response to fish cue indicating fish predation. The availability of food allowed the animals to stay in the cold hypolimnion. However, at the low food level (0.1 mg C l−1), which is an additional constraint on fitness, Daphnia moved away from the cold hypolimnion. Poor food condition resulted in strong migrant Daphnia to cease migration and remain in the upper warmer water layer. Although temperature is known to be a more important cost factor of DVM than food, our results clearly show that this is only true as long as food is available. It becomes clear that food availability is controlling the direction of vertical positioning when daphnids experience a dilemma between optimising temperature and food condition while being exposed to fish cue. Then they overlook the predation risk. Thus, the optimal habitat choice of Daphnia appears to be a function of several variables including temperature, food levels and fish predation.

Quick discrimination of heavy metal resistant bacterial populations using infrared spectroscopy coupled with chemometrics.

Lead and cadmium are frequently encountered heavy metals in industrially polluted areas. Many heavy metal resistant bacterial strains have a high biosorption capacity and thus are good candidates for the removal of toxic metals from the environment. However, as of yet there is no accurate method for discrimination of highly adaptive bacterial strains among the populations present in a given habitat. In this study, we aimed to find distinguishing molecular features of lead and cadmium resistant bacteria using Attenuated Total Reflectance-Fourier Transformed Infrared (ATR-FT-IR) spectroscopy and chemometric approaches. Our results demonstrated that both control and metal exposed E. coli and S. aureus strains could be successfully discriminated from each other using hierarchical cluster and principal component analysis methods. Moreover, we found that lead exposed bacterial strains could be successfully discriminated from cadmium exposed ones with a high heterogeneity value. These clear discriminations can be described by the ability of a bacterium to change its metabolism in terms of the content and structure of cellular macromolecules under heavy metal stress. In our case, cadmium and lead-induced genetic response systems in bacteria caused remarkable alterations in overall cellular metabolism. Bacteria deal with a heavy metal stress by altering nucleic acid methylations and lipid and protein synthesis. Heavy metal burden led to the development of relevant metabolic changes in proteins, lipids, and nucleic acids of the resistant bacteria described in this study. Our approach showed that infrared spectra obtained via ATR-FT-IR spectroscopy coupled with chemometric analysis can be utilized for rapid, low-cost, informative, reliable, and operator-independent discrimination of resistant bacterial populations.

Rapid classification of heavy metal-exposed freshwater bacteria by infrared spectroscopy coupled with chemometrics using supervised method

Rapid, cost-effective, sensitive and accurate methodologies to classify bacteria are still in the process of development. The major drawbacks of standard microbiological, molecular and immunological techniques call for the possible usage of infrared (IR) spectroscopy based supervised chemometric techniques. Previous applications of IR based chemometric methods have demonstrated outstanding findings in the classification of bacteria. Therefore, we have exploited an IR spectroscopy based chemometrics using supervised method namely Soft Independent Modeling of Class Analogy (SIMCA) technique for the first time to classify heavy metal-exposed bacteria to be used in the selection of suitable bacteria to evaluate their potential for environmental cleanup applications. Herein, we present the powerful differentiation and classification of laboratory strains (Escherichia coli and Staphylococcus aureus) and environmental isolates (Gordonia sp. and Microbacterium oxydans) of bacteria exposed to growth inhibitory concentrations of silver (Ag), cadmium (Cd) and lead (Pb). Our results demonstrated that SIMCA was able to differentiate all heavy metal-exposed and control groups from each other with 95% confidence level. Correct identification of randomly chosen test samples in their corresponding groups and high model distances between the classes were also achieved. We report, for the first time, the success of IR spectroscopy coupled with supervised chemometric technique SIMCA in classification of different bacteria under a given treatment.

Aspects of silver tolerance in bacteria: infrared spectral changes and epigenetic clues

In this study, the molecular profile changes leading to the adaptation of bacteria to survive and grow at inhibitory silver concentration were explored. The profile obtained through infrared (IR)-based measurements indicated extensive changes in all biomolecular components, which were supported by chemometric techniques. The changes in biomolecular profile were prominent, including nucleic acids. The changes in nucleic acid region (1350-950 cm-1 ) were encountered as a clue for conformational change in DNA. Further analysis of DNA by IR spectroscopy revealed changes in the backbone and sugar conformations. Moreover, Enzyme-Linked Immunosorbent Assay-based measurements of DNA methylation levels were performed to see if epigenetic mechanisms are in operation during bacterial adaptation to this environmental challenge. The results indicated a notable demethylation in Escherichia coli and methylation in Staphylococcus aureus likely to be associated with their elaborate adaptation process to sustain survival and growth.

The effects of repeated antibiotic administration to juvenile BALB/c mice on the microbiota status and animal behavior at the adult age

Recent studies carried on germ –free (GF) animal models suggest that the gut microbiota (GM) may play a role in the regulation of anxiety, mood, and cognitive abilities such as memory and learning processes. Consistently, any treatment disturbing the gut microbiota, including the overuse of antibiotics, may influence the brain functions and impact behavior. In the present study, to address this issue, two wide-spectrum antibiotics (ampicillin and cefoperazone, 1 g/l) were repeatedly applied throughout a 6-week period to initially 21-day-old male BALB/c mice. Antibiotics were administered separately or in a mixed fashion. On the completion of the antibiotic treatment, all mice were subjected to the behavioral tests. The serum levels of corticosterone and brain-derived neurotropic factor (BDNF) were assessed. Gut microbiota profiles were obtained by using denaturing gradient gel electrophoresis system, DGGE, from fecal samples. Ampicillin had a greater impact on both, gut microbiota composition and mice behavior compared to cefoperazone. All antibiotic-treated groups manifested a decrease in the locomotor activity and reduced recognition memory. However, the ampicillin-treated groups showed a higher anxiety level as assessed by the open field and the elevated plus maze tests and an increased immobility (behavioral despair) in the forced swim test. Obtained results evidently show that in mice, a repeated antibiotic treatment applied during adolescence, parallel to the changes in GM, affects locomotor activity, affective behavior and cognitive skills in young adults with ampicillin specifically enhancing anxiety- and depressive-like responses. Lower levels of serum BDNF were not associated with cognitive impairment but with changes in affective-like behaviors. Repeated administration of neither ampicillin nor cefoperazone affected basal serum corticosterone levels. This is one of the few studies demonstrating changes in a behavioral phenotype of young-adult subjects who were previously exposed to a repeated antibiotic treatment.

Comparison of microbially induced calcium carbonate precipitation eligibility using sporosarcina pasteurii and bacillus licheniformis on two different sands

Abstract The use of biological means for ground improvement have become popular, which generally works through the process called microbially-induced calcium carbonate precipitation (MICP). Many studies indicate successful application of MICP based improvement with multiple bacteria and on several soils. Given the proven performance of MICP, this study aims to examine the MICP process by comparing the calcium carbonate precipitation ability of widely studied bacteria, i.e., Sporosarcina pasteurii and relatively under-recognized bacteria, i.e., Bacillus licheniformis to outline the formation success. For this purpose, two different sands were tested for observing precipitation behavior using a series of syringe tests. Furthermore, the effect of concentration and inclusion of calcium chloride for nutrition of bacteria, saturation with water, and hybrid use of two bacteria were investigated in some tests for diversification. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive x-ray spectroscopy (EDS) were used for the interpretation of results. Results indicated that Sporosarcina pasteurii had performed superior over Bacillus licheniformis when achieving calcium carbonate precipitation in tests for both sands. In addition, many intriguing SEM images contributed to the literature of MICP monitoring, highlighting the effects of the variables investigated.