Effects of antibiotic-induced resistance on the growth, survival ability and virulence of Salmonella enterica.

Salmonella enterica is an important foodborne pathogen that constitutes a major health hazard. The emergence and aggravation of antibiotic-resistant Salmonella has drawn attention widely around the world. Conducting a risk assessment of antibiotic-resistant foodborne pathogens throughout the food chain is a pressing requirement for ensuring food safety. The growth, survival capability, and virulence of antibiotic-resistant Salmonella represent crucial biological characteristics that play an important role in microbial risk assessment. In this study, eight antibiotic-sensitive S. enterica strains were induced by Ampicillin (Amp) and Ciprofloxacin (CIP), respectively, and AMP-resistant and CIP-resistant mutants were obtained. The growth characteristics under different temperatures (25, 30, 35 °C), viability after exposure to heat (55, 57.5, 60 °C) and acid (HCl, pH = 3.0), the virulence potential (adhesion and invasion to Caco-2 cells, biofilm formation and motility) and the lethality in a model species (Galleria mellonella) were evaluated and compared for S. enterica strains before and after antibiotic exposure. The induction by AMP and CIP are likely to promote cross-antibiotic resistance to their antibiotic classes, ß-lactams and quinolones, as well as some compound antibiotics. It was observed that generally the antibiotic-induction-resistant strains showed decreased growth ability and lower heat resistance, although the differences were not significant at all the conditions tested. The AMP-resistant strains were significantly less acid resistance than the sensitive and the CIP-resistant ones, while exhibiting increased biofilm formation ability. In general, the antibiotic-induced resistance did not significantly affect the motility, adherence, or invasion ability of Caco-2 cells. However, CIP-resistant strains displayed lower lethality in G. mellonella infection, whereas AMP-resistant strains did not, and even two strains improved lethality. The study of the biological characteristics of antibiotic-resistant S. enterica is essential in better understanding the microbial risks to both the food chain and human health, thereby facilitating a more accurate risk assessment.

Dual-Gene Isothermal Amplification Coupled with Lateral Flow Strip for On-Site Accurate Detection of E. coli O157:H7 in Food Samples.

On-site field detection of E. coli O157:H7 in food samples is of utmost importance, since it causes a series of foodborne diseases due to infections-associated ready-to-eat foods. Due to the instrument-free nature, recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) is well-suited for such goal. However, the high genomic similarity of different E. coli serotypes adds difficulty to accurate differentiation of E. coli O157:H7 from others. Dual-gene analysis could significantly improve the serotype selectivity, but will further aggravate the RPA artifacts. To address such issue, here we proposed a protocol of dual-gene RPA-LFA, in which the target amplicons were selectively recognized by peptide nucleic acid (PNA) and T7 exonuclease (TeaPNA), thus eliminating false-positives in LFA readout. Adapting rfbEO157 and fliCH7 genes as the targets, dual-gene RPA-TeaPNA-LFA was demonstrated to be selective for E. coli O157:H7 over other E. coli serotypes and common foodborne bacteria. The minimum detection concentration was 10 copies/µL for the genomic DNA (∼300 cfu/mL E. coli O157:H7), and 0.24 cfu/mL E. coli O157:H7 in food samples after 5 h bacterial preculture. For lettuce samples contaminated with E. coli O157:H7 (single-blind), the sensitivity and specificity of the proposed method were 85% and 100%, respectively. Using DNA releaser for fast genomic DNA extraction, the assay time could be reduced to ∼1 h, which is appealing for on-site food monitoring.

Facile monitoring of meat freshness with a self-constructed photosensitization colorimetric instrument.

Total volatile basic nitrogen (TVB-N) produced from the decomposition of amino acids is an important indicator for meat freshness. Various pH-sensitive colorimetric films have been incorporated as intelligent packaging for meat freshness during food transportation. However, methods and instruments capable of on-site end-point detection of meat freshness are still needed for places that provide raw meat without packaging. Herein, based on amine-induced pH change that led to decreased color output of the 3,3',5,5'-tetramethylbenzidine (TMB)-based photosensitization colorimetric assay, a simple yet convenient instrument employing colorimetric indicator paper (CIP) was constructed for facile monitoring of meat freshness. Owing to the background color provided by the photosensitizer erythrosine (2',4',5',7'-tetraiodofluorescein, TIF), the color changed from blue to pink upon amine adsorption. A bespoke cellphone App was employed for image capture and color analysis of the CIP for freshness monitoring. The analytical results of amine (released from meat during storage) by the proposed method agreed well with those by a standard Conway dish method. In addition, the whole analytical process could be completed in about 5 min. The developed instrument may be potentially useful for on-site monitoring of meat freshness.

Inactivation and Subsequent Growth Kinetics of Listeria monocytogenes After Various Mild Bactericidal Treatments.

This study was carried out to investigate the effects of mild heat, lactic acid, benzalkonium chloride and nisin treatments on the inactivation, sublethal injury, and subsequent growth of Listeria monocytogenes. Results showed that the Bigelow model successfully described the thermal inactivation kinetics, while the Log-linear model with tail consistently offered the most accurate fit to LA, BC, and nisin inactivation curves of cells. Differential plating indicated that percentage of sublethal injury for nisin treated cells was significantly higher than that for the other three treatments. Compared to non-treated cells, significant extension of lag time was observed for all treated cells. The longer exposures to heat treatment contributed to the extended lag time of the survivors. While for LA, BC and nisin treated cells, the longest lag time was not observed at the most severe treatment conditions. The correlation analysis of sublethal injury percentage on the duration of lag time revealed that only heat treatment showed the significant correlation. Overall, the lag time analysis could evaluate a wide range of bacterial injury. Lag time of treated cells was significantly influenced by stress treatments and temperatures of recovery, however, there were not any significant changes in the maximum specific growth rate between treated and non-treated cells under isothermal recovery conditions. The information generated from this study is valuable for utilizing intervention strategies in the elimination or growth inhibition of L. monocytogenes.

Recombinase Polymerase Amplification Coupled with a Photosensitization Colorimetric Assay for Fast Salmonella spp. Testing.

Salmonella spp. is one of the most serious foodborne pathogens causing millions of infection cases annually, especially in resource-limited areas. The standard culture method (2-3 days) and current nucleic acid amplification-based testing are not suitable for on-site testing in rural areas with heavy Salmonella spp. burden. Here, we developed a colorimetric recombinase polymerase amplification (RPA) method for fast and sensitive Salmonella spp. testing in 1 h. Specifically, the invA gene from the genomic DNA of Salmonella spp. was amplified isothermally to produce double-stranded DNA (dsDNA) amplicons, which were directly quantified by a photosensitization colorimetric assay. The proposed method offered the lowest detectable concentration of 5 × 103 colony-forming units/mL (cfu/mL), which is much lower than that of ELISA (105-107 cfu/mL). The detectable limit could be further pushed down to 3 cfu/mL upon coupling with bacteria pre-enrichment for 6 h. Analysis of synthetic milk samples confirmed the high precision (90%) and specificity (95%) of the method for Salmonella spp. testing. Moreover, use of a DNA releaser could further simplify the whole testing operation. Because RPA features low-temperature amplification (25-42 °C) without the need for specific instruments and the dsDNA-based photosensitization colorimetric assay served as a simple and facile readout for RPA, our method thus allows fast and low-cost Salmonella spp. testing in food samples.

Phosphorescent inner filter effect-based sensing of xanthine oxidase and its inhibitors with Mn-doped ZnS quantum dots.

Overexpression and crystallization of uric acid have been recognized as the course of hyperuricemia and gout, which is produced via xanthine oxidase (XOD)-catalyzed oxidation of xanthine. Therefore, the medicinal therapy of hyperuricemia and gout is majorly based on the inhibition of the XOD enzymatic pathway. The spectroscopic nature of xanthine and uric acid, namely both absorption (near the ultraviolet region) and emission (non-fluorescent) characteristics, hinders optical assay development for XOD analysis. Therefore, the state-of-the-art analysis of XOD and the screening of XOD inhibitors are majorly based on chromatography. Here, we found the near ultraviolet absorption of uric acid overlapped well with the absorption of a large bandgap semiconductor quantum dots, ZnS. On the other hand, the intrinsic weak fluorescence of ZnS QDs can be substantially improved via transition metal ion doping. Therefore, herein, we developed an inner filter effect-based assay for XOD analysis and inhibitor screening with Mn-doped ZnS QDs. The phosphorescence of Mn-doped ZnS QDs could be quenched by uric acid generated from xanthine catabolism by XOD, leading to the phosphorescence turn-off detection of XOD with a limit of detection (3σ) of 0.02 U L-1. Furthermore, the existence of XOD inhibitors could inhibit the XOD enzymatic reaction, resulting in weakened phosphorescence quenching. Therefore, the proposed assay could also be explored for the facile screening analysis of XOD inhibitors, which is important for the potential medicinal therapy of hyperuricemia and gout.

Ratiometric Phosphorescent Probe for Thallium in Serum, Water, and Soil Samples Based on Long-Lived, Spectrally Resolved, Mn-Doped ZnSe Quantum Dots and Carbon Dots.

Thallium (Tl) is an extremely toxic heavy metal and exists in very low concentrations in the environment, but its sensing is largely underexplored as compared to its neighboring elements in the periodic table (especially mercury and lead). In this work, we developed a ratiometric phosphorescent nanoprobe for thallium detection based on Mn-doped ZnSe quantum dots (QDs) and water-soluble carbon dots (C-dots). Upon excitation with 360 nm, Mn-doped ZnSe QDs and C-dots can emit long-lived and spectrally resolved phosphorescence at 580 and 440 nm, respectively. In the presence of thallium, the phosphorescence emission from Mn-doped ZnSe QDs could be selectively quenched, while that from C-dots retained unchanged. Therefore, a ratiometric phosphorescent probe was thus developed, which can eliminate the potential influence from both background fluorescence and other analyte-independent external environment factors. Several other heavy metal ions caused interferences to thallium detection but could be efficiently masked with EDTA. The proposed method offered a detection limit of 1 µg/L, which is among the most sensitive probes ever reported. Successful application of this method for thallium detection in biological serum as well as in environmental water and soil samples was demonstrated.

Phosphorescent sensing of Cr3+ with protein-functionalized Mn-doped ZnS quantum dots.

It was found that the phosphorescence from denatured bovine serum albumin (dBSA)-capped Mn-doped ZnS QDs could be selectively quenched by Cr(3+), and a phosphorescent probe for Cr(3+) was thus developed. Based on phosphorescence decay as well as calculations of the relative energies of QDs and Cr(3+), the mechanism for phosphorescent quenching was preliminarily ascribed to electron transfer from photo-excited Mn-doped ZnS QDs to Cr(3+). Under the optimal conditions, good linear Stern-Volmer quenching was obtained for Cr(3+) in the range of 10 to 300 nM. The limit of detection of this phosphorescence probe (3 nM) was 1 to 2 orders of magnitude lower than those of previously reported nanosensors, owing to the effective elimination of background fluorescence and scattering from the sample matrix. The analytical potential of the proposed probe was evaluated through determination of Cr(3+) in water samples, with spike-recoveries ranging from 95 to 106%.

Interaction between room temperature ionic liquid [bmim]BF4 and DNA investigated by electrochemical micromethod.

Ionic liquids (ILs) as a kind of novel green solvent are being widely used in various researches related to the life sciences and chemistry, which demands the knowledge of interaction between ILs and biomacromolecules. However, the almost completely inert optical, electric, thermal properties of ILs make it difficult to directly obtain information about the interactions. Herein, by using a hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) as a model, the electrostatic interaction between ILs and calf thymus DNA (ctDNA) was investigated by a surface electrochemical micromethod. A convenient and simple method was established to obtain the thermodynamic and kinetic information about the DNA-IL interaction only with microscale sample consumption. The quantitative thermodynamic and kinetic parameters about the interaction of [bmim]BF4 and ctDNA, such as the binding constant (K), the Gibbs energy of surface binding (DeltaGb), and the dissociation rate constant (k), were obtained for the first time.

Flow-focusing generation of monodisperse water droplets wrapped by ionic liquid on microfluidic chips: from plug to sphere.

Generating droplets via microfluidic chips is a promising technology in microanalysis and microsynthesis. To realize room-temperature ionic liquid (IL)-water two-phase studies in microscale, a water-immiscible IL was employed as the continuous phase for the first time to wrap water droplets (either plugs or spheres) on flow-focusing microfluidic chips. The IL, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), could wet both hydrophilic and hydrophobic channel surfaces because of its dual role of hydrophilicity/hydrophobicity and extremely high viscosity, thus offering the possibility of wrapping water droplets in totally hydrophilic (THI), moderately hydrophilic (MHI), and hydrophobic (HO) channels. The droplet shape could be tuned from plug to sphere, with the volume from 6.3 nL to 65 pL, by adding an orifice in the focusing region, rendering the hydrophilic channel surface hydrophobic, and suppressing the Uw/UIL ratio below 1.0. Three different breakup processes were defined and clarified, in which the sub-steady breakup and steady breakup were essential for the formation of plugs and spheric droplets, respectively. The influences of channel hydrophilicity/hydrophobicity on droplet formation were carefully studied by evaluating the wetting abilities of water and IL on different surfaces. The superiority of IL over water in wetting hydrophobic surface led to the tendency of forming small, spheric aqueous droplets in the hydrophobic channel. This IL-favored droplet-based system represented a high efficiency in water/IL extraction, in which rhodamine 6G was extracted from aqueous droplets to [BMIM][PF6] in the hydrophobic orifice-included (HO-OI) channel in 0.51 s.