Genotyping of Campylobacter jejuni and prediction tools of its antimicrobial resistance.

Although Campylobacter jejuni is the pathogen responsible for the most common foodborne illness, tracing of the infection source remains challenging due to its highly variable genome. Therefore, one of the aim of the study was to compare three genotyping methods (MLST, PFGE, and mP-BIT) to determine the most effective genotyping tool. C. jejuni strains were divided into 4 clusters based on strain similarity in the cgMLST dendrogram. Subsequently, the dendrograms of the 3 tested methods were compared to determine the accuracy of each method compared to the reference cgMLST method. Moreover, a cost-benefit analysis has showed that MLST had the highest inverse discrimination index (97%) and required less workflow, time, fewer consumables, and low bacterial sample quantity. PFGE was shown to be obsolete both because of its low discriminatory power and the complexity of the procedure. Similarly, mP­BIT showed low separation results, which was compensated by its high availability. Therefore, our data showed that MLST is the optimal tool for genotyping C. jejuni. Another aim was to compare the antimicrobial resistance to ciprofloxacin, erythromycin, and tetracycline in C. jejuni strains isolated from human, water, air, food, and animal samples by two gene sequence-based prediction methods and to compare them with the actual susceptibility of C. jejuni strains using the disc diffusion method. Both tools, ResFinder and RGI, synchronously predict the antimicrobial susceptibility of C. jejuni and either can be used.

Absolute energy levels in nanodiamonds of different origins and surface chemistries.

Nanodiamonds (NDs) are versatile, broadly available nanomaterials with a set of features highly attractive for applications from biology over energy harvesting to quantum technologies. Via synthesis and surface chemistry, NDs can be tuned from the sub-micron to the single-digit size, from conductive to insulating, from hydrophobic to hydrophilic, and from positively to negatively charged surface by simple annealing processes. Such ND diversity makes it difficult to understand and take advantage of their electronic properties. Here we present a systematic correlated study of structural and electronic properties of NDs with different origins and surface terminations. The absolute energy level diagrams are obtained by the combination of optical (UV-vis) and photoelectron (UPS) spectroscopies, Kelvin probe measurements, and energy-resolved electrochemical impedance spectroscopy (ER-EIS). The energy levels and density of states in the bandgap of NDs are correlated with the surface chemistry and structure characterized by FTIR and Raman spectroscopy. We show profound differences in energy band shifts (by up to 3 eV), Fermi level position (from p-type to n-type), electron affinity (from +0.5 eV to -2.2 eV), optical band gap (5.2 eV to 5.5 eV), band gap states (tail or mid-gap), and electrical conductivity depending on the high-pressure, high-temperature and detonation origin of NDs as well as on the effects of NDs' oxidation, hydrogenation, sp2/sp3 carbon phases and surface adsorbates. These data are fundamental for understanding and designing NDs' optoelectrochemical functional mechanisms in diverse application areas.

Size-Dependent Thermal Stability and Optical Properties of Ultra-Small Nanodiamonds Synthesized under High Pressure.

Diamond properties down to the quantum-size region are still poorly understood. High-pressure high-temperature (HPHT) synthesis from chloroadamantane molecules allows precise control of nanodiamond size. Thermal stability and optical properties of nanodiamonds with sizes spanning range from <1 to 8 nm are investigated. It is shown that the existing hypothesis about enhanced thermal stability of nanodiamonds smaller than 2 nm is incorrect. The most striking feature in IR absorption of these samples is the appearance of an enhanced transmission band near the diamond Raman mode (1332 cm-1). Following the previously proposed explanation, we attribute this phenomenon to the Fano effect caused by resonance of the diamond Raman mode with continuum of conductive surface states. We assume that these surface states may be formed by reconstruction of broken bonds on the nanodiamond surfaces. This effect is also responsible for the observed asymmetry of Raman scattering peak. The mechanism of nanodiamond formation in HPHT synthesis is proposed, explaining peculiarities of their structure and properties.

Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni.

Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species, naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81-176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated ([Formula: see text]luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

The effect of environmental conditions on the occurrence of Campylobacter jejuni and Campylobacter coli in wastewater and surface waters.

AIMS: The purpose of the study was to evaluate the occurrence of Campylobacter jejuni and Campylobacter coli in the aquatic environment based on the water origin, seasonality and physico-chemical properties. METHODS AND RESULTS: The occurrence of C. jejuni and C. coli was determined in waste (29) or surface (56) waters in four different seasons. The air and water temperatures were measured during sampling and chemical analyses of water samples for ammonium, chloride, chlorine, nitrite, nitrate, phosphate and iron were performed. The thermotolerant Campylobacter spp. were more frequently detected in wastewater (59%; 17 positive samples) compared to surface water (38%; 21 positive samples), with the highest rate in autumn (67% of samples positive) and with a higher C. coli occurrence than C. jejuni (31% vs. 26%). Ammonium (above 0.2 mg/L) and chloride ion concentrations (above 60 mg/L) favour C. jejuni. Similarly, C. coli occurrence in water was supported by ammonium (above 0.2 mg/L), chloride (above 60 mg/L) and in addition by phosphate ion concentrations (below 0.7 mg/L). CONCLUSIONS: Campylobacter presence in water is influenced by physico-chemical parameters such as concentrations of ammonium and chloride ions. SIGNIFICANCE AND IMPACT OF THE STUDY: Water environment is an alternative source of Campylobacter. The concentration of ammonium and chloride ions can be used as a basis for successful prediction of the potential occurrence of C. jejuni and C. coli in wastewater and surface water in future.

Waterborne Isolates of Campylobacter jejuni Are Able to Develop Aerotolerance, Survive Exposure to Low Temperature, and Interact With Acanthamoeba polyphaga.

Campylobacter jejuni is regarded as the leading cause of bacterial gastroenteritis around the world. Even though it is generally considered to be a sensitive microaerobic pathogen, it is able to survive in the environment outside of the intestinal tract of the host. This study aimed to assess the impact of selected environmental parameters on the survival of 14 C. jejuni isolates of different origins, including 12 water isolates. The isolates were tested for their antibiotic resistance, their ability to survive at low temperature (7°C), develop aerotolerance, and to interact with the potential protozoan host Acanthamoeba polyphaga. The antibiotic susceptibility was determined by standard disk diffusion according to EUCAST. Out of the 14 isolates, 8 were resistant to ciprofloxacin (CIP) and 5 to tetracycline (TET), while only one isolate was resistant to erythromycin (ERY). Five isolates were resistant to two different antibiotic classes. Tetracycline resistance was only observed in isolates isolated from wastewater and a clinical sample. Further, the isolates were tested for their survival at 7°C under both aerobic and microaerobic conditions using standard culture methods. The results showed that under microaerobic conditions, all isolates maintained their cultivability for 4 weeks without a significant decrease in the numbers of bacteria and variation between the isolates. However, significant differences were observed under aerobic conditions (AC). The incubation led to a decrease in the number of cultivable cells, with complete loss of cultivability after 2 weeks (one water isolate), 3 weeks (7 isolates), or 4 weeks of incubation (6 isolates). Further, all isolates were studied for their ability to develop aerotolerance by repetitive subcultivation under microaerobic and subsequently AC. Surprisingly, all isolates were able to adapt and grow under AC. As the last step, 5 isolates were selected to evaluate a potential protective effect provided by A. polyphaga. The cocultivation of isolates with the amoeba resulted in the survival of about 40% of cells treated with an otherwise lethal dose of gentamicin. In summary, C. jejuni is able to adapt and survive in a potentially detrimental environment for a prolonged period of time, which emphasizes the role of the environmental transmission route in the spread of campylobacteriosis.

Laser-Induced Modification of Hydrogenated Detonation Nanodiamonds in Ethanol.

Apart from the frequently used high-temperature annealing of detonation nanodiamonds (DNDs) in an inert environment, laser irradiation of DNDs in a liquid can be effectively used for onion-like carbon (OLC) formation. Here, we used fully de-aggregated hydrogenated DNDs (H-DNDs) dispersed in ethanol, which were irradiated for up to 60 min using a 532 nm NdYAG laser with an energy of 150 mJ in a pulse (5 J/cm2) at a pulse duration of 10 ns and a repetition rate of 10 Hz. We investigated the DND surface chemistry, zeta potential, and structure as a function of laser irradiation time. Infrared spectroscopy revealed a monotonical decrease in the C-Hx band intensities and an increase of the C-O and C=O features. Transmission electron microscopy (TEM) revealed the formation of OLC, as well as a gradual loss of nanoparticle character, with increasing irradiation time. Surprisingly, for samples irradiated up to 40 min, the typical and unchanged DND Raman spectrum was recovered after their annealing in air at 450 °C for 300 min. This finding indicates the inhomogeneous sp3 to sp2 carbon transformation during laser irradiation, as well as the insensitivity of DND Raman spectra to surface chemistry, size, and transient structural changes.

Adhesion, Biofilm Formation, and luxS Sequencing of Campylobacter jejuni Isolated From Water in the Czech Republic.

The microaerophilic pathogen Campylobacter jejuni is a leading bacterial cause of human gastroenteritis in developed countries. Even though it has a reputation as a fastidious organism, C. jejuni is widespread and can be easily isolated from various animals, food, and environmental sources. It is suggested that an ability to form biofilms is probably necessary for the survival of C. jejuni under harsh environmental conditions. The first step required for successful biofilm formation is adhesion to a suitable surface. Therefore, in this work, the degree of adhesion was evaluated, followed by characterization and quantification of biofilms using confocal laser scanning microscopy (CLSM). A total of 15 isolates of C. jejuni were used in the experiments (12 isolates from surface and waste waters, 1 human clinical, 1 food and 1 ACTT BAA-2151 collection strain, all samples originated from the Czech Republic). Regardless of the sample origin, all C. jejuni isolates were able to adhere to the polystyrene surface within 30 min, with the number of attached cells increasing with the time of incubation. The resulting data showed that all isolates were able to form complex voluminous biofilms after 24 h of cultivation. The average amount of biovolume ranged from 3.59 × 106 µm3 to 17.50 × 106 µm3 in isolates obtained from different sources of water, 16.79 × 106 µm3 in the food isolate and 10.92 × 106 µm3 in the collection strain. However, the highest amount of biomass was produced by the human clinical isolate (25.48 × 106 µm3). Similar to the quantity, the architecture of the biofilms also differed, from a rugged flat monolayer of cells to large clustered structures. Further, all isolates were tested for the presence of the luxS gene, as the luxS/AI-2 (autoinducer-2) quorum sensing pathway has been previously connected with enhanced biofilm formation. Two isolates originated from surface waters did not possess the luxS gene. These isolates formed thinner and sparser biofilms lacking the presence of significant clusters. However, the ability to adhere to the surface was preserved. The sequencing of the luxS-containing fragments shown a high similarity of the luxS gene among the isolates.