Pathogenic Enteric Viruses and Microbial Indicators during Secondary Treatment of Municipal Wastewater.

Pathogenic enteric viruses are responsible for a wide range of infections in humans, with diverse symptoms. Raw and partially treated wastewaters are major sources of environmental contamination with enteric viruses. We monitored a municipal secondary wastewater treatment plant (New Orleans, LA) on a monthly basis for norovirus (NoV) GI and GII and enterovirus serotypes using multiplex reverse transcription-quantitative PCR (RT-qPCR) and microbial indicators of fecal contamination using standard plating methods. Densities of indicator bacteria (enterococci, fecal coliforms, and Escherichia coli) did not show monthly or seasonal patterns. Norovirus GII was more abundant than GI and, along with enterovirus serotypes, increased in influent during fall and spring. The highest NoV GI density in influent was in the fall, reaching an average of 4.0 log10 genomic copies/100 ml. Norovirus GI removal (0.95 log10) was lower than that for GII, enterovirus serotypes, and male-specific coliphages (1.48 log10) or for indicator bacteria (4.36 log10), suggesting higher resistance of viruses to treatment. Male-specific coliphages correlated with NoV GII densities in influent and effluent (r = 0.48 and 0.76, respectively) and monthly removal, indicating that male-specific coliphages can be more reliable than indicator bacteria to monitor norovirus GII load and microbial removal. Dominant norovirus genotypes were classified into three GI genotypes (GI.1, GI.3, and GI.4) and four GII genotypes (GII.3, GII.4, GII.13, and GII.21), dominated by GI.1 and GII.4 strains. Some of the seasonal and temporal patterns we observed in the pathogenic enteric viruses were different from those of epidemiological observations.

Minimizing human infection from Escherichia coli O157:H7 using GUMBOS.

OBJECTIVES: Reduction in faecal shedding of Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) in food-producing animals is a viable strategy to minimize human disease initiated by exposure to these microorganisms. To this end, an intervention strategy involving the electrostatic hybridization of two commonly used anti-infective agents for veterinary practice (i.e. chlorhexidine and ampicillin) was evaluated to curtail EHEC-transmitted disease from ruminant sources. Chlorhexidine di-ampicillin is a novel group of uniform material based on organic salts (GUMBOS) with inherent in vitro antibacterial activity that comes from its parent antimicrobial ions, chlorhexidine and ampicillin. METHODS: Antibacterial activities for chlorhexidine diacetate, sodium ampicillin, chlorhexidine di-ampicillin and stoichiometrically equivalent 1 : 2 chlorhexidine diacetate : sodium ampicillin were assessed using the serial 2-fold dilution method and time-kill studies against seven isolates of E. coli O157:H7 and one non-pathogenic E. coli 25922. Further studies to investigate synergistic interactions of reacted and stoichiometrically equivalent unreacted antimicrobial agents at MICs and possible mechanisms were also investigated. RESULTS: Synergism and in vitro antibacterial activities against EHEC were observed in this study, which suggests chlorhexidine di-ampicillin could be a useful reagent in reducing EHEC transmission and minimizing EHEC-associated infections. Likewise, chlorhexidine di-ampicillin reduced HeLa cell toxicity as compared with chlorhexidine diacetate or the stoichiometric combination of antimicrobial agents. Further results suggest that the mechanisms of action of chlorhexidine di-ampicillin and chlorhexidine diacetate against E. coli O157:H7 are similar. CONCLUSIONS: Reacting antimicrobial GUMBOS as indicated in this study may enhance the approach to current combination drug therapeutic strategies for EHEC disease control and prevention.

Effectiveness of Aqueous Chlorine Dioxide in Minimizing Food Safety Risk Associated with Salmonella, E. coli O157:H7, and Listeria monocytogenes on Sweet Potatoes.

Sodium hypochlorite (NaOCl) is a commonly used sanitizer in the produce industry despite its limited effectiveness against contaminated human pathogens in fresh produce. Aqueous chlorine dioxide (ClO2) is an alternative sanitizer offering a greater oxidizing potency with greater efficacy in reducing a large number of microorganisms. We investigated the effect of aqueous chlorine dioxide treatment against human pathogens, Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes seeded on sweet potatoes. Sweet potatoes were spot inoculated (4.2 to 5.7 log CFU/cm2) with multi-strain cocktails of Salmonella spp., E. coli O157:H7, and L. monocytogenes and treated for 10-30 min with 5 ppm aqueous ClO2 or water. Aqueous ClO2 treatment was significantly (p < 0.05) effective in reducing Salmonella with a reduction of 2.14 log CFU/cm2 within 20 min compared to 1.44 log CFU/cm2 for water treatment. Similar results were observed for L. monocytogenes with a 1.98 log CFU/cm2 reduction compared to 0.49 log CFU/cm2 reduction observed after 30 min treatment with aqueous ClO2 the water respectively. The maximum reduction in E. coli O157: H7 reached 2.1 Log CFU/cm2 after 20 min of treatment with aqueous ClO2. The level of the pathogens in ClO2 wash solutions, after the treatment, was below the detectable limit. While in the water wash solutions, the pathogens' populations ranged from 3.47 to 4.63 log CFU/mL. Our study indicates that aqueous ClO2 is highly effective in controlling cross-contamination during postharvest washing of sweet potatoes.

Hyaluronic Acid-Cellulose Composites as Patches for Minimizing Bacterial Infections.

A facile method was used to synthesize biocomposites containing differing ratios of hyaluronic acid (HA) and cellulose (CEL). Based on the properties of the individual polymers, the resultant composite materials may have potentially great wound care properties. In the method outlined here, 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), a simple ionic liquid, was used as the sole solvent without chemical modifiers to dissolve the biopolymers at ratios of 1:1 and 2:1 HA to CEL. This method was completely recyclable since the ionic liquid, [Bmim][Cl], can be recovered. Results from spectroscopic measurements [Fourier transform infrared (FT-IR) and X-ray diffraction (XRD)] confirm the interaction between HA and CEL. Scanning electron microscopy (SEM) images reflect differing biopolymer ratios and the resulting impact on the texture and porosity of these composite materials. The composites exhibited high swelling capacity in various media. These composites were also drug-loaded to examine drug release properties for greater potential in combating Staphylococcus aureus infections.

Edible chitosan films on ready-to-eat roast beef for the control of Listeria monocytogenes.

The use of chitosan as an edible film was evaluated for its antimicrobial activity against Listeria monocytogenes (LM) on the surface of ready-to-eat (RTE) roast beef. L. monocytogenes, decimally diluted to give an initial inoculation of >6.50logCFU/g, was inoculated onto the surface of RTE roast beef cubes, and air-dried. The samples were dipped into chitosan (high or low molecular weights) solutions dissolved with acetic or lactic acid at 0.5% (w/v) or 1% (w/v) then bagged and refrigerated at 4 degrees C. The bacterial counts were determined on days 0, 7, 14, 21, and 28. The samples were spread plated onto modified Oxford agar plates and incubated at 37 degrees C for 48h. An initial 6.50logCFU/g of L. monocytogenes inoculated onto the surface of the non-coated RTE roast beef increased too >10logCFU/g by day 28. On day 14, L. monocytogenes counts were significantly different for all the chitosan-coated samples from the control counts by 2-3logCFU/g and remained significantly different on day 28. Our results have shown that the acetic acid chitosan coating were more effective in reducing L. monocytogenes counts than the lactic acid chitosan coating. Our study indicated that chitosan coatings could be used to control L. monocytogenes on the surface of RTE roast beef.

Consumer Acceptance, Emotional Response, and Purchase Intent of Rib-Eye Steaks from Grass-Fed Steers, and Effects of Health Benefit Information on Consumer Perception.

Sensory acceptability, purchase intent (PI), consumer emotional profile, and fatty acid profile of rib-eye steaks from four different biological types of grass-fed cattle (Angus, Brangus, Pineywoods, and Holstein) were evaluated. The effects of health benefit information (HBI) pertaining to grass-fed beef on the overall liking (OL), emotional profile, and PI were also studied. Consumers rated steaks from the two nontraditional beef biological types (Pineywoods and Holstein) equally or higher than conventional biological types (Angus and Brangus) in several hedonic (9-point scale) categories. Mean OL scores were 6.1 for Pineywoods and 6.3 for Holstein steaks, versus 5.5 for Angus and 6.0 for Brangus. Hedonic attributes, especially OL and liking of beef flavor, accounted for most of the differences between treatments based on Wilks' Lambda Multivariate Analysis of Variance (MANOVA) and descriptive discriminant analysis. After knowledge of HBI, OL of all treatments increased, particularly Angus with OL mean score significantly increasing from 5.5 to 6.2. Positive PI increased significantly for Angus (from 48% to 65.33%), Brangus (from 50.67% to 66.67%), and Holstein (from 61.33% to 76%) steaks. Angus steaks, which initially received the lowest beef flavor liking (5.1) showed the most improvement for OL after HBI. Likewise, emotional profiles were improved after HBI, particularly the "Interested" emotion with a 0.5 unit increased. PRACTICAL APPLICATION: Providing consumers with health benefit information (HBI) can enhance acceptability of grass-fed beef. Fatty acid analyses of steaks from four biological types of steers showed favorable n-6/n-3 ratios. Although some sensory attributes of cooked rib-eye steaks were scored differently among biological types, knowledge of grass-feeding and healthier fatty acids increased overall liking and purchase intent (PI) across products. Emotional profiles improved after HBI was given to consumers, with some emotions being significantly related to PI odds ratios. This research demonstrated the usefulness of HBI in improving hedonic perceptions of grass-fed steaks and the importance of consumer emotions in predicting PI.

Acidified sodium chlorite treatment for inhibition of Listeria monocytogenes growth on the surface of cooked roast beef.

The effects of acidified sodium chlorite (ASC) against Listeria monocytogenes on the surface of cooked roast beef were investigated. L. monocytogenes, strain V7, serotype 1/2a, was inoculated at numbers of 6.0 log CFU/g onto 5-g cubes of cooked regular or spicy roast beef. The samples were allowed to air dry for 1 h. The cooked roast beef samples were dipped into ASC or sprayed with ASC solutions of 250, 500, 750, or 1,000 ppm, then placed in bags with or without a vacuum and refrigerated at 4 degrees C. L. monocytogenes counts were determined after 0, 7, 14, 21, and 28 days of storage by spread plating roast beef samples onto Oxford agar plates that were incubated at 37 degrees C for 48 h. At day 28, the number of L. monocytogenes on the > or = 500 ppm ASC-treated spicy roast beef samples had count reductions that were >4.0 log CFU/g, whereas the same concentrations of ASC-treated regular roast beef samples had approximately a 2.5 log CFU/g reduction in L. monocytogenes counts when compared with the untreated samples. No significant differences (P > 0.05) were observed in L. monocytogenes counts between the vacuum- or nonvacuum-packaged ASC-treated cooked roast beef samples. Sensory evaluation showed no significant differences (P > 0.05) between ASC-treated and untreated roast beef. ASC can be used as a processing aid in the form of a dip or spray treatment to control L. monocytogenes on the surface of cooked roast beef.

Differential killing activity of cetylpyridinium chloride with or without bacto neutralizing buffer quench against firmly adhered Salmonella gaminara and Shigella sonnei on cut lettuce stored at 4 degrees C.

Cetylpyridinium chloride (CPC) activity was quenched with Bacto neutralizing buffer on inoculated cut iceberg lettuce. This protocol permitted comparison of the numbers of Salmonella Gaminara- or Shigella sonnei-inoculated cells on lettuce that survived 1 min of CPC treatment. Cut lettuce was inoculated with about 6 log of Salmonella or 9 log of Shigella and stored in Whirl-Pak bags at 4 degrees C for up to 4 days. Loosely adhered pathogen cells were washed off before CPC treatment. Firmly adhered cells of Salmonella Gaminara or S. sonnei on cut iceberg lettuce survived treatment with CPC even at the 0.4% CPC level if the CPC activity was quenched after 1 min by adding Bacto neutralizing buffer. The results confirm that there is extended killing activity of residual CPC against Salmonella Gaminara or S. sonnei if the residual CPC remaining in contact with the lettuce after the initial 1-min wash is not quenched. The CPC treatment was useful in reducing the numbers of these target pathogens on lettuce.

Transmission electron microscopy study of enterohemorrhagic Escherichia coli O157:H7 in apple tissue.

We investigated the ability of enterohemorrhagic Escherichia coli O157:H7 to spread in wounded apple tissue by transmission electron microscopy. Red Delicious apples were wounded with an artist knife (7 mm depth) and either inoculated with 10 microl per wound of decimally diluted E. coli O157:H7 or submerged into E. coli O157:H7 suspended in sterile distilled water and then stored at 37 degrees C for 24 h. Transmission electron microscopy showed E. coli O157:H7 formed bacterial aggregates near the apple cell walls, and single cells were in close proximity to the apple cell wall surfaces and to plasma membranes. E. coli O157:H7 presence caused degradation of plasma membranes and release of the cytoplasm contents of the apple cortical cells into the central vacuole. Apple tissue turgor pressure tests showed that the apple cells infected with E. coli O157:H7 isolates were more likely to rupture than the control noninoculated apple cells. E. coli O157:H7 cells grown in apple tissue showed the formation of granules and vesicles within the bacterial cytoplasma and separation of the plasma membranes. Our study shows that E. coli O157:H7 can grow and survive in the apple tissue environment by causing degradation of the apple cellular components.

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast.

Noroviruses are the most common causative agent of viral gastroenteritis in humans, and are responsible for major foodborne illnesses in the United States. Filter-feeding molluscan shellfish exposed to sewage-contaminated waters bioaccumulate viruses, and if consumed raw, transmit the viruses to humans and cause illness. We investigated the occurrence of norovirus GI and GII and microbial indicators of fecal contamination in the eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast (January to November of 2013). Microbial indicators (aerobic plate count, enterococci, fecal coliforms, Escherichia coli, male-specific coliphages, and somatic coliphages) were detected at the densities lower than public health concerns. Only one oyster sample was positive for norovirus GII at 3.5 ± 0.2 log10 genomic equivalent copies/g digestive tissues. A stool specimen obtained from an infected individual associated with a norovirus outbreak and the suspected oysters (Cameron Parish, La., area 30, January 2013) were also analyzed. The norovirus strain in the stool belonged to GII.4 Sydney; however, the oysters were negative and could not be linked. In general, no temporal trend was observed in the microbial indicators. Low correlation among bacterial indicators was observed in oysters. Strongest correlations among microbial indicators were observed between enterococci and fecal coliforms (r = 0.63) and between enterococci and E. coli (r = 0.64) in water (P < 0.05); however, weak correlations were found in oysters (r < 0.45) and between oysters and harvest water (r ≤ 0.36, P > 0.05). Our results emphasize the need for regular monitoring of pathogenic viruses in commercial oyster harvesting areas to reduce the risks of viral gastroenteritis incidences.

Cross-Cultural Consumer Acceptability and Purchase Intent of Forage-Finished Rib-Eye Steaks.

UNLABELLED: Rib-eye steaks, from 3 forage-finished systems (S1, S2, and S3) and 1 commercial steak (C), either cooked by 1-sided-grilling or 2-sided-grilling, were evaluated for sensory acceptability [overall appearance (ORA) and overall appearance of fat (OAF) for raw steaks; overall appearance (OCA), overall beef aroma (OBA), overall beef flavor (OBF), juiciness, tenderness and overall liking (OL) for cooked steaks] and purchase intent by Hispanic, Asian and U.S. consumers. They also indicated preferred degree of doneness and cooking methods. Cross-cultural differences in preferences and consumer acceptability of rib-eye steaks were observed. Grilling was the most preferred cooking method. Hispanics and Asians preferred medium and/or medium well, while U.S. consumers preferred medium and/or medium rare. For cooked steaks, the population effect was significant for all sensory attributes; Asians generally scored lower than did Hispanics and U.S. consumers. C and S3 generally had higher scores for all sensory attributes across 3 populations. Purchase intent for all forage-finished steaks was higher for Hispanics and U.S. consumers compared to Asians (50.0% to 77.8% compared with 43.2% to 65.9%). Attributes influencing purchase intent of forage-finished steaks differed among populations: tenderness (odds ratio = 1.4) for Hispanics, OCA (odds ratio = 1.5) for Asians, and OBF (odds ratio = 1.3) for U.S. consumers. Overall, this study demonstrated that the type of forage-finished system and ethnic differences influenced sensory acceptability and purchase intent of forage-finished rib-eye steaks. PRACTICAL APPLICATION: Demand for forage-finished beef has increased worldwide due to its potential health benefits. Little is known regarding the cross-cultural effects on the consumer acceptance of forage-finished beef. We evaluated sensory acceptance and purchase intent of raw and cooked forage-finished rib-eye steaks using Hispanic, Asian, and U.S. (White and African American) populations. This study demonstrated that consumer acceptability and purchase intent of forage-finished rib-eye steaks differed among Hispanic, Asian, and U.S. populations. This information can be used to guide development and marketing strategies for beef products.

Antimicrobial biocompatible bioscaffolds for orthopaedic implants.

Nationally, nearly 1.5 million patients in the USA suffer from ailments requiring bone grafts and hip and other joint replacements. Infections following internal fixation in orthopaedic trauma can cause osteomyelitis in 22-66% of cases and, if uncontrolled, the mortality rate can be as high as 2%. We characterize a procedure for the synthesis of antimicrobial and biocompatible poly-l-lactic acid (PLLA) and poly-ethyleneglycol (PEG) bioscaffolds designed to degrade and absorb at a controlled rate. The bioscaffold architecture aims to provide a suitable substrate for the controlled release of silver nanoparticles (SNPs) to reduce bacterial growth and to aid the proliferation of human adipose-derived stem cells (hASCs) for tissue-engineering applications. The fabricated bioscaffolds were characterized by scanning transmission microscope (SEM) and it showed that the addition of tncreasing concentrations of SNPs results in the formation of dendritic porous channels perpendicular to the axis of precipitation. The antimicrobial properties of these porous bioscaffolds were tested according to a modified ISO 22196 standard across varying concentrations of biomass-mediated SNPs to determine an efficacious antimicrobial concentration. The bioscaffolds reduced the Staphylococcus aureus and Escherichia coli viable colony-forming units by 98.85% and 99.9%, respectively, at an antimicrobial SNPs concentration of 2000 ppm. Human ASCs were seeded on bioscaffolds and cultured in vitro for 20 days to study the effect of SNPs concentration on the viability of cells. SEM analysis and the metabolic activity-based fluorescent dye, AlamarBlue®, demonstrated the growth of cells on the efficacious antimicrobial bioscaffolds. The biocompatibility of in vitro leached silver, quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES), proved non-cytotoxic when tested against hASCs, as evaluated by MTT assay.

Design, synthesis, and biological evaluation of ß-lactam antibiotic-based imidazolium- and pyridinium-type ionic liquids.

We herein report the preparation and investigation of antibacterial activity of biocidal ionic liquids (ILs) consisting of cationic imidazolium or pyridinium and an anionic ß-lactam antibiotic. The antibacterial properties were quantified by measuring the minimum inhibitory concentration and minimum bactericidal concentration against Escherichia coli O157:H7, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecium. In general, the ILs had improved antibacterial activity than their parent materials, whether individually or in combination. In 83% of the experiments, the ampicillin ILs (Amp-ILs) had better antibacterial activities than their quaternary halide parent materials, whereas in 92% of the experiments, Amp-ILs outperformed the commercially available sodium ampicillin salt. Amp-ILs had up to 43 times improved antibacterial activity than sodium ampicillin. Overall, when normalized for ampicillin content, ILs had greater antimicrobial activity against E. coli O157:H7, K. pneumoniae, S. aureus, and E. faecium than sodium ampicillin alone.

Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters.

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC > or = 16 microg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.