Mediator-less immunodetection with voltage-controlled intrinsic amplification for ultrasensitive and rapid detection of microorganism pathogens

A mediator-less immunodetection method for microorganisms is realized by incorporating the newly developed field-effect enzymatic detection (FEED) technique with the conventional electrochemical immunosensing approach. The gating voltage of FEED facilitates the transduction of electrical signal through the bulky immune complex so that the detection does not rely on the use of mediators or other diffusional substances. The voltage-controlled intrinsic amplification provided by the detection system allows detection in low-concentration samples without target pre-enrichment, leading to ultrasensitive and rapid detection. The detection approach is demonstrated with E. coliO157:H7, a model microorganism, in milk with an estimated detection limit of 20 CFU mL−1 (where CFU is a colony-forming unit) without performing sample pre-enrichment and centrifugation of sample followed by the resuspension of the pellet in a buffer solution, resulting in a significantly shortened assay time of 67 min. Optimizing the gating voltage resulted in the detection of 12 CFU mL−1 of the bacterium in milk. The novel detection approach can be used as a detection platform for ultrasensitive, specific and rapid detection of microorganism pathogens

Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 104 and 105 colony forming units/mL or 0.1–0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification

Depuration as a method to reduce Vibrio vulnificus populations in live Crassostrea virginica oysters

Vibrio vulnificus is a foodborne bacterial pathogen associated with raw oyster consumption. Shellfish depuration for 48 hours is a dynamic process where coliform bacteria are purged; however, this process is ineffective against V. vulnificus. The current study investigated the use of prolonged two-week depuration on V. vulnificus populations in Gulf Coast oysters. The study evaluated the impact of prolonged depuration on V. vulnificus fatty acid profile change and the ability to survive in simulated gastric fluid. Oyster depuration in seawater (10 or 22oC, 14 days) reduced V. vulnificus counts, but not to non-detectable level, indicating close ecological relationship between the pathogen and mollusk. Greatest V. vulnificus count reductions were seen in 12 ppt 10°C seawater (2.7 log10 CFU/g) and in 20 ppt 22°C seawater (2.8 logs). Mesophilic vibrios dominated the overall microflora of freshly harvested oysters, while refrigeration selected for psychrotrophic bacteria. Depuration at 22°C retained dominance of mesophilic vibrios, including pathogenic species. Although aerobic plate counts were lower in 22°C depurated oysters (5.0 logs vs 6.0 logs), depuration at 10°C had little to no advantage over 22°C in terms of vibrio population reduction. Use of prolonged depuration remains economically questionable since this method failed to completely eliminate V. vulnificus. Starved V. vulnificus behavior in artificial seawater showed that low temperature (4oC) and high seawater salinity (35 ppt) contributed to pathogen population reduction. Starved V. vulnificus did not adjust membrane fluidity to storage temperature within the investigated time frame. However, a significant fatty acid switch from C18:1w7c to C18:1w6c by double bond relocation was observed. The relocation was faster at ambient temperatures compared to refrigerated temperatures. The majority of V. vulnificus foodborne infections occur during warm summer months. Vibrio vulnificus ATCC 27562 was significantly less resistant (3.7 min D-value) to simulated gastric fluid (pH 4.0) after 7-day storage at 4oC compared to the control (7.8 min D-value). Therefore, greater gastric fluid sensitivity of the pathogen may occur in winter-harvested oysters and may partially explain the low number of winter outbreaks

Mechanism of Synergistic Inhibition of Listeria monocytogenes Growth by Lactic Acid, Monolaurin, and Nisin▿

The combined lactic acid, monolaurin, and nisin effects on time-to-detection (optical density at 600 nm) extension were greater (P < 0.05) than any single or paired combination effect, which demonstrates a synergistic interaction among the antimicrobials. Monolaurin exposure caused C12:0 cell membrane incorporation. Lactic acid caused increased monolaurin C12:0 membrane incorporation, while nisin had no influence. We postulate that lactic acid-enhanced monolaurin C12:0 incorporation into the cell membrane increased membrane fluidity resulting in increased nisin activity

INFLUENCE OF LOW TEMPERATURE STORAGE AND SALINITY ON FATTY ACID PROFILE AND INACTIVATION BY SIMULATED GASTRIC JUICE OF HUMAN PATHOGENIC BACTERIUM VIBRIO VULNIFICUS

Under starvation conditions in artificial seawater, cells of V vulnificus did not adjust their membrane fluidity to storage temperature within the investigated time frame. However, a significant switch (p&lt;0.05) from C18:1m7c into С 18:1 обе by double bond relocation was observed. The relocation occurred faster at ambient temperatures compared to refrigerated temperatures. It is generally known that majority of V. vulnificus associated infections occur during summer hot months. Vibrio vulnificus ATCC 27562 was significantly less resistant to simulated gastric fluid (pH 4.0) after 7-day storage at 4 °С compared to the control, with D-values of 3.7 and 7.8 minutes, respectively. Therefore, higher sensitivity of the pathogen to the gastric fluid in winter harvested oysters may also impact the low number of outbreaks