James L. McEvoy

Local Modulation of Host pH by Colletotrichum Species as a Mechanism to Increase Virulence

The phytopathogenic fungus Colletotrichum gloeosporioides produces one pectate lyase (PL) that is a key virulence factor in disease development. During growth of C. gloeosporioides, Colletotrichum acutatum, and Colletotrichum coccodes in acidified yeast extract medium, the fungus secreted ammonia and increased the medium pH. Ammonia accumulation and the consequent pH change increased as a function of initial pH and buffer capacity of the medium. PL secretion by C. gloeosporioides correspondingly increased as the pH of the medium increased. The C. gloeosporioides pelB gene-disrupted mutant was able to increase ammonia accumulation and pH of the media similarly to the wild-type isolate. C. gloeosporioides in avocado, C. coccodes in tomato, and C. acutatum in apple showed ammonia accumulation in the infected area where pH increased to 7.5 to 8 and PL activity is optima. In nonhost interactions where C. gloeosporioides was inoculated in apples, the addition of ammonia-releasing compounds significantly enhanced pathogenicity to levels similar to those caused by the compatible C. acutatum-apple interaction. The results therefore suggest the importance of ammonia secretion as a virulence factor, enhancing environmental pH and pathogenicity of the Colletotrichum species.

Efficacy of Sanitizers To Inactivate Escherichia coli O157:H7 on Fresh-Cut Carrot Shreds under Simulated Process Water Conditions†

Chlorine is widely used as a sanitizer to maintain the microbial quality and safety of fresh-cut produce; however, chlorine treatment lacks efficacy on pathogen reduction, especially when the fresh-cut processing water contains heavy organic loads. A more efficacious sanitizer that can tolerate the commercial processing conditions is needed to maintain microbial safety of fresh-cut produce. This study evaluated the efficacy of Escherichia coli O157:H7 reduction on fresh-cut carrots using new and traditional sanitizers with tap water and fresh-cut processing water scenarios. Fresh-cut carrot shreds inoculated with E. coli O157:H7 were washed in sanitizer solutions including 200 ppm chlorine, citric acid-based sanitizer (Pro-San), 80 ppm peroxyacetic acid-based sanitizer (Tsunami 100), and 1,000 ppm acidified sodium chlorite (SANOVA) prepared in fresh tap water or simulated processing water with a chemical oxygen demand level of approximately 3,500 mg/liter. Samples were packaged and stored at 5 degrees C. Microbial analyses performed at days 0, 7, and 14 indicate that the organic load in the process water significantly affected the efficacy of chlorine on pathogen removal and was especially evident on samples tested during storage. Acidified sodium chlorite provided a strong pathogen reduction even under process water conditions with up to a 5.25-log reduction when compared with the no-wash control. E. coli O157:H7 was not recovered on acidified sodium chlorite-treated samples during the entire 14 days of storage, even following an enrichment step. These results suggest that acidified sodium chlorite holds considerable promise as an alternative sanitizer of fresh-cut produce.

Fate of Escherichia coli O157:H7 in the presence of indigenous microorganisms on commercially packaged baby spinach, as impacted by storage temperature and time.

This study investigated the effect of storage temperature and time on the survival and growth of Escherichia coli O157:H7, the growth of indigenous microorganisms, and the changes in product quality of packaged baby spinach. Commercial packages of spinach within 2 days of processing were cut open at one end, sprayed with fine mists of E. coli O157:H7 inoculum, resealed, and then stored at 1, 5, 8, and 12 degrees C for 12 days until their labeled best-if-used-by dates. Microbial enumeration and product quality evaluation were conducted on day(s) 0, 3, 6, 9, and 12 postinoculation. Spinach held at 12 degrees C supported significant (P < 0.001) E. coli O157:H7 growth, with a 1.0-log CFU/g increase within 3 days postinoculation, which was followed by additional growth during continued storage. E. coli O157:H7 grew slowly when held at 8 degrees C, with a significant (P < 0.01) level of growth reached after 6 days of storage. However, on products held at 1 and 5 degrees C, E. coli O157:H7 populations declined significantly (P < 0.01 and P < 0.001, respectively) within 3 days of storage. Aerobic mesophilic bacteria, psychrotrophic bacteria, and yeast and mold populations increased significantly at all storage temperatures, with more growth on products held at elevated temperatures. Product quality scores remained high within the first 6 days of storage, with a sharp decline noted on samples held at 12 degrees C on day 9. Results suggest that E. coli O157:H7 can grow significantly on commercially packaged spinach held at 8 degrees C or above before significant product quality deterioration occurs.

Characterization of Salmonella isolates from retail foods based on serotyping, pulse field gel electrophoresis, antibiotic resistance and other phenotypic properties.

Sixteen Salmonella strains isolated from a variety of foods during 2000 and 2003 by the Florida State Department of Agriculture were characterized by various genotypic and phenotypic tests. Among 16 isolates, 15 different serotypes were identified. Pulse-field gel electrophoresis (PFGE) fingerprinting profiles obtained using restriction endonucleases XbaI and BlnI revealed that 16 Salmonella isolates were genetically diverse with 16 unique PFGE patterns. The PFGE pattern of eight isolates matched with the CDC/FDA data base of previous outbreaks and clinical isolates indicating their potential to cause disease. With the exception of isolates obtained from alligator meat (tetracycline resistant) and orange juice (chloramphenicol and sulfisoxazole resistant), the remainder of the isolates were susceptible to the panel of 15 antimicrobials tested. Molecular subtyping was further complimented by a variety of phenotypic tests such as acid-tolerance, Caco-2 cell invasion and biofilm formation which have often been used as a gauge of virulence and infection potential of Salmonella isolates. The induced acid tolerance level of the isolate obtained from orange juice was not significantly different from the laboratory reference strain S. enterica serovar Typhimurium SL1344. Six isolates exhibited very low levels of constitutive acid-tolerance, of which four isolates failed to infect differentiated Caco-2 cells. Although all isolates formed biofilms, there was no clear relation between the ability to form biofilms, infect differentiated Caco-2 cells and induce acid-tolerance. This study indicated that different serotypes of Salmonella were present in a variety of retail foods and exhibited diverse phenotypic characteristics.

Assessment of Sodium Hypochlorite and Acidified Sodium Chlorite as Antimicrobial Agents to Inhibit Growth of Escherichia coli O157:H7 and Natural Microflora on Shredded Carrots

Abstract Acidified sodium chlorite (ASC) is an alternative to chlorine in reducing microbial populations to maintain food quality and safety on fresh-cut produce. However the most effective concentration for microbial reduction on shredded carrots is unknown. In this study the influence of tap water, sodium hypochlorite (SH; 200 mg · L−1) and acidified sodium chlorite (ASC; 100,250,500 and 1,000 mg·L−1) washes on natural microflora, and survival and growth of Escherichia coli O157:H7 inoculated onto shredded carrots, was determined after treat-ment and 7 and 14 days of storage. The carrots were stored under passive modified atmosphere at 5°C. While a tap water wash did not reduce growth of E. coli O157:H7, total plate count or yeast and molds, spoilage and pathogenic microorganisms were reduced by two logs by using SH or ASC at 200 and 100 mg·L−1, respectively. ASC at concentrations above 100 mg·L−1 was very effective in reducing microbial growth by 6 logs. During storage, total mesophilic growth increased in samples washed with tap water, SH or ASC at 100 and 250 mg·L−1. However, shredded carrots washed with ASC at 1000 mg·L−1 did not show any microbial growth even after 14 days of storage at 5°C.