Jane M. Caldwell

One-step synthesis of silver nanoparticle-filled nylon 6 nanofibers and their antibacterial properties

A novel and facile one-step approach to in situ synthesize silver nanoparticle-filled nylon 6 nanofibers by electrospinning is reported. The method does not need post-treatments and can be carried out at ambient conditions without using additional chemicals. It employs the electrospinning solvent as a reducing agent for in situ conversion of AgNO3 into silver nanoparticles during the solution preparation. The resultant silver nanoparticle-filled nylon 6 hybrid nanofibers show an excellent fibrous structure (fiber diameter at 50–150 nm), with narrow size 2–4 nm silver nanoparticles uniformly dispersed throughout the nylon 6 matrix. DSC analysis shows that the in situ incorporation of silver nanoparticles increased the Tg and crystallinity of the resultant nanofibers. These silver nanoparticle-filled nylon 6 nanofibers exhibit a steady and long-lasting silver ion release behavior, and robust antibacterial activity against both Gram-positive B. cereus and Gram-negative E. coli microorganisms.

Multifunctional ZnO/Nylon 6 nanofiber mats by an electrospinning-electrospraying hybrid process for use in protective applications.

Abstract ZnO/Nylon 6 nanofiber mats were prepared by an electrospinning–electrospraying hybrid process in which ZnO nanoparticles were dispersed on the surface of Nylon 6 nanofibers without becoming completely embedded. The prepared ZnO/Nylon 6 nanofiber mats were evaluated for their abilities to kill bacteria or inhibit their growth and to catalytically detoxify chemicals. Results showed that these ZnO/Nylon 6 nanofiber mats had excellent antibacterial efficiency (99.99%) against both the Gram-negative Escherichia coli and Gram-positive Bacillus cereus bacteria. In addition, they exhibited good detoxifying efficiency (95%) against paraoxon, a simulant of highly toxic chemicals. ZnO/Nylon 6 nanofiber mats were also deposited onto nylon/cotton woven fabrics and the nanofiber mats did not significantly affect the moisture vapor transmission rates and air permeability values of the fabrics. Therefore, ZnO/Nylon 6 nanofiber mats prepared by the electrospinning–electrospraying hybrid process are promising material candidates for protective applications.

Domestic wastewater influent profiling using mitochondrial real-time PCR for source tracking animal contamination

Real-time PCR amplifying mammalian and avian mitochondrial DNA (mtDNA) was developed to characterize domestic and light industrial wastewater influent from two municipal wastewater treatment facilities (WWTF) over a 24-week period. Influent samples were assayed with species-specific primers and dual-labeled probes for human, bovine, swine, dog, cat, Canada goose and white-tailed deer to detect and quantify eukaryotic mtDNA contributors to local municipal wastewaters. Human (mean=9.6 x 10(4) copies/ml) and dog (mean=5.3 x 10(2) copies/ml) mtDNA were detected in all 24 samples (12 samples/site). Bovine and swine mtDNA were detected sporadically and at lower levels than human mtDNA, means=3.0 x 10(4) and 9.5 x 10(2) copies/ml, respectively. Domestic cat, Canada goose and white-tailed deer were detected only once in 24 samples. Mitochondrial DNA concentrations were compared to other bacterial, chemical and spectrophotometric parameters. Human mtDNA was positively correlated with ammonia concentration (P=0.01) and initial OD(600) reading (P=0.02) at one WWTF. Bovine mtDNA was positively correlated with biological oxygen demand (BOD) (P=0.02), final DNA concentration (P=0.03), initial and final humic acid concentrations (P=0.01, P=0.01), and final OD(600) (P=0.03) at one WWTF and total suspended solids (TSS) (P=0.04, P=0.09) at both facilities. Fecal coliforms were not positively or negatively correlated with mtDNA concentrations of any species assayed. For source tracking purposes, a combination of human (10(5) copies/ml) and dog mtDNA signal (10(2) copies/ml) could be indicative of municipal domestic wastewater contamination of environmental waters.

Survival of Escherichia coli O157:H7 in cucumber fermentation brines.

UNLABELLED Bacterial pathogens have been reported on fresh cucumbers and other vegetables used for commercial fermentation. The Food and Drug Administration currently has a 5-log reduction standard for E. coli O157:H7 and other vegetative pathogens in acidified pickle products. For fermented vegetables, which are acid foods, there is little data documenting the conditions needed to kill acid resistant pathogens. To address this knowledge gap, we obtained 10 different cucumber fermentation brines at different stages of fermentation from 5 domestic commercial plants. Cucumber brines were used to represent vegetable fermentations because cabbage and other vegetables may have inhibitory compounds that may affect survival. The 5-log reduction times for E. coli O157:H7 strains in the commercial brines were found to be positively correlated with brine pH, and ranged from 3 to 24 d for pH values of 3.2 to 4.6, respectively. In a laboratory cucumber juice medium that had been previously fermented with Lactobacillus plantarum or Leuconostoc mesenteroides (pH 3.9), a 5-log reduction was achieved within 1 to 16 d depending on pH, acid concentration, and temperature. During competitive growth at 30 °C in the presence of L. plantarum or L. mesenteroides in cucumber juice, E. coli O157:H7 cell numbers were reduced to below the level of detection within 2 to 3 d. These data may be used to aid manufacturers of fermented vegetable products determine safe production practices based on fermentation pH and temperature. PRACTICAL APPLICATION   Disease causing strains of the bacterium E. coli may be present on fresh vegetables. Our investigation determined the time needed to kill E. coli in cucumber fermentation brines and how E. coli strains are killed in competition with naturally present lactic acid bacteria. Our results showed how brine pH and other brine conditions affected the killing of E. coli strains. These data can be used by producers of fermented vegetable products to help assure consumer safety.

Mitochondrial DNA as Source Tracking Markers of Fecal Contamination

Guidelines for water-quality monitoring have traditionally focused on the use of bacterial indicators. However, efforts to effectively mitigate fecal contamination necessitate greater clarity in source recognition. Host (mammalian and avian) epithelial cells are shed in the gut lumen and expelled in feces. These cells have multiple numbers of mitochondria, an organelle with its own genome, containing species-specific DNA sequences. These properties make mitochondrial DNA sequences (mtDNA) excellent molecular targets as they are host-specific and robust. This chapter describes the development of molecular methods such as PCR, qPCR, PCR with consensus primers, and DNA microarrays to detect and quantify mtDNA in effluents, influents, and environmental surface waters. These assays represent a paradigm shift in source tracking by detecting DNA from the host rather than its fecal bacterial population. Future development to increase the sensitivity of the assays and ease sample processing of large volumes is warranted. Contamination by nonfecal sources such as skin, hair, and sputum of swimmers needs to be evaluated in the context of providing data for source tracking, i.e., presence of human activity impacting the site. The significance of meat carryover in human feces, waste from kitchen garbage disposals and abattoir or industrial manufacturing requires further study to assess their impact on species-specific source tracking.

Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant‐Derived, Low‐Acid Foods, and Biomaterials

Cycle threshold (Ct) increase, quantifying plant-derived DNA fragmentation, was evaluated for its utility as a time-temperature integrator. This novel approach to monitoring thermal processing of fresh, plant-based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time-temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low-acid product and simple linear regression, Ct value was highly correlated to time-temperature treatment (R(2) = 0.87); the logarithmic reduction (log CFU/mL) of the spore-forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R(2) = 0.87); and cumulative F-value (min) in a canned retort process (R(2) = 0.88), all comparisons conducted at 121 °C. D121 and z-values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D121 and z-values for a 174-bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof-of-concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low-acid plant products.

Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods

Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products.

Stable Transmission of Borrelia burgdorferi Sensu Stricto on the Outer Banks of North Carolina

The spirochaete (Borrelia burgdorferi) associated with Lyme disease was detected in questing ticks and rodents during a period of 18 years, 1991–2009, at five locations on the Outer Banks of North Carolina. The black‐legged tick (Ixodes scapularis) was collected at varied intervals between 1991 and 2009 and examined for B. burgdorferi. The white‐footed mouse (Peromyscus leucopus), house mouse (Mus musculus) marsh rice rat (Oryzomys palustris), marsh rabbit (Sylvilagus palustris), eastern cottontail (Sylvilagus floridanus) and six‐lined racerunner (Cnemidophorus sexlineatus) were live‐trapped, and their tissues cultured to isolate spirochaetes. Borrelia burgdorferi isolates were obtained from questing adult I. scapularis and engorged I. scapularis removed from P. leucopus, O. palustris and S. floridanus. The prevalence of B. burgdorferi infection was variable at different times and sites ranging from 7 to 14% of examined questing I. scapularis. Mitochondrial (16S) rRNA gene phylogenetic analysis from 65 adult I. scapularis identified 12 haplotypes in two major clades. Nine haplotypes were associated with northern/Midwestern I. scapularis populations and three with southern I. scapularis populations. Sixteen isolates obtained from tick hosts in 2005 were confirmed to be B. burgdorferi by amplifying and sequencing of 16S rRNA and 5S‐23S intergenic spacer fragments. The sequences had 98–99% identity to B. burgdorferi sensu stricto strains B31, JD1 and M11p. Taken together, these studies indicate that B. burgdorferi sensu stricto is endemic in questing I. scapularis and mammalian tick hosts on the Outer Banks of North Carolina.

Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.