Joshua B. Gurtler

Stress, Sublethal Injury, Resuscitation, and Virulence of Bacterial Foodborne Pathogens

Environmental stress and food preservation methods (e.g., heating, chilling, acidity, and alkalinity) are known to induce adaptive responses within the bacterial cell. Microorganisms that survive a given stress often gain resistance to that stress or other stresses via cross-protection. The physiological state of a bacterium is an important consideration when studying its response to food preservation techniques. This article reviews the various definitions of injury and stress, sublethal injury of bacteria, stresses that cause this injury, stress adaptation, cellular repair and response mechanisms, the role of reactive oxygen species in bacterial injury and resuscitation, and the potential for cross-protection and enhanced virulence as a result of various stress conditions.

Cronobacter sakazakii in foods and factors affecting its survival, growth, and inactivation

Cronobacter sakazakii has been isolated from a wide range of environmental sources and from several foods of animal and plant origin. While infections caused by C. sakazakii have predominantly involved neonates and infants, its presence on or in foods other than powdered infant formula raises concern about the safety risks these foods pose to immunocompromised consumers. We have done a series of studies to better understand the survival and growth characteristics of C. sakazakii in infant formula, infant cereal, fresh-cut produce, and juices made from fresh produce. Over a 12-month storage period, the pathogen survived better in dried formula and cereal at low a(w) (0.25-0.30) than at high a(w) (0.69-0.82) and at 4 degrees C compared to 30 degrees C. C. sakazakii grows in formulas and cereals reconstituted with water or milk and held at 12-30 degrees C. The composition of formulas or cereals does not markedly affect the rate of growth. C. sakazakii grows well on fresh-cut apple, cantaloupe, watermelon, cabbage, carrot, cucumber, lettuce, and tomato at 25 degrees C and in some types of produce at 12 degrees C. Treatment of fresh fruits and vegetables with sanitizers such as chlorine, chlorine dioxide, and a peroxyacetic acid-based solution causes reductions of 1.6-5.4 log CFU/apple, tomato, and lettuce. Cells of C. sakazakii in biofilms formed on stainless steel and enteral feeding tubes or dried on the surface of stainless steel have increased resistance to disinfectants. Death of cells in biofilms is affected by atmospheric relative humidity. These studies have contributed to a better understanding of the behavior of C. sakazakii in and on foods and on food-contact surfaces, thereby enabling the development of more effective strategies and interventions for its control.

Selection of surrogate bacteria in place of E. coli O157:H7 and Salmonella Typhimurium for pulsed electric field treatment of orange juice.

Pulsed electric field (PEF) technology has been used for the inactivation of microorganisms and to prevent flavor loss in liquid foods and beverages in place of thermal pasteurization. When used to pasteurize orange juice, PEF may prevent loss of volatile sensory attributes. Enterohemorrhagic E. coli O157:H7 (EHEC), two strains of Salmonella Typhimurium, and twenty strains of non-pathogenic bacteria were screened for inactivation in orange juice by PEF at 22 and 20kV/cm at 45 and 55 degrees C, respectively. Higher populations of both salmonellae were inactivated (2.81 and 3.54 log CFU/ml) at 55 degrees C, in comparison with the reduction of EHEC (2.22 log). When tested under the same conditions, inactivation of EHEC was slightly greater than that of a non-pathogenic E. coli (NPEC) ATCC 35218 (2.02 log). NPEC was further tested as a surrogate for EHEC by comparing inactivation kinetics at 45, 50 and 55 degrees C at field strengths of between 7.86 and 32.55kV/cm. Statistical comparison of revealed that EHEC and NPEC inactivation curves were homogeneous at outlet temperatures of 45 and 50 degrees C; however, EHEC was slightly more sensitive to PEF than the surrogate NPEC at 55 degrees C. The higher PEF resistance of non-pathogenic E. coli 35218 at 55 degrees C may provide a desirable margin of safety when used in pilot plant challenge studies in place of E. coli O157:H7.

Inactivation of Salmonella in liquid egg albumen by antimicrobial bottle coatings infused with allyl isothiocyanate, nisin and zinc oxide nanoparticles

Aims:  To develop an antimicrobial bottle coating effective at inhibiting the growth of Salmonella in liquid egg albumen (egg white) and reduce the risk of human Salmonellosis.

Inactivation of Salmonella on whole cantaloupe by application of an antimicrobial coating containing chitosan and allyl isothiocyanate.

This study investigated the antimicrobial effect of a chitosan coating+allyl isothiocyanate (AIT) and nisin against Salmonella on whole fresh cantaloupes. Cantaloupes were inoculated with a cocktail of three Salmonella strains and treated with chitosan, chitosan+AIT, chitosan+nisin, and chitosan+AIT+nisin coatings. With AIT concentrations increasing from 10 to 60 μl/ml, the antibacterial effects of coating treatments against Salmonella increased. Chitosan coatings with 60 μl/ml AIT (chitosan+60AIT) reduced more than 5 log₁₀ CFU/cm² of Salmonella. The addition of nisin to the chitosan-AIT coating synergistically increased the antibacterial effect; coatings with nisin (25 mg/ml or 25,000 IU/ml)+30 μl/ml AIT resulted in a 4.8 log₁₀ reduction of Salmonella. The chitosan+60AIT coating significantly (p<0.05) reduced populations of native bacteria on cantaloupes to ca. 2 log₁₀ CFU/cm² during the first 6 days and populations remained unchanged through day 14 at 10 °C. The same coating treatment completely inactivated mold and yeast on cantaloupe at day 1 and no regrowth occurred even up to 14 days of storage. Scanning electron microscopy revealed that cell membrane damage and leakage of intercellular components occurred as a result of the chitosan-AIT coating treatments. No visual changes in overall appearance and color of cantaloupe rind and flesh due to coating treatments were observed. These results indicate that the application of an antimicrobial coating may be an effective method for decontamination of cantaloupes.

Survival of Enterobacter sakazakii in powdered infant formula as affected by composition, water activity, and temperature.

A study was done to determine survival characteristics of Enterobacter sakazakii in four milk-based and two soybean-based powdered infant formulas. A 10-strain mixture of E. sakazakii was inoculated into the six infant formulas at water activity (aw) 0.25 to 0.30, 0.31 to 0.33, and 0.43 to 0.50 to give low (0.80 log CFU/g) and high (4.66 to 4.86 log CFU/g) populations. At an initial population of 0.80 log CFU/g, E. sakazakii was detected by enrichment in six of six, four of six, and one of six formulas stored for 12 months at 4, 21, and 30 degrees C, respectively. In four of six formulas at aw values of 0.25 to 0.30, initially high populations decreased significantly (P < or = 0.05), although by less than 1 log CFU/g, within 6 months at 4 degrees C. Populations decreased significantly in all formulas in the aw range of 0.25 to 0.50 during storage for 1 month at 21 or 30 degrees C and again between 1 and 6 months in most formulas. Significant reductions occurred between 6 and 12 months in some formulas. At all storage temperatures, reductions in populations tended to be greater in formulas at aw 0.43 to 0.50 than in formulas at aw 0.25 to 0.30. The rate of inactivation of E. sakazakii in formulas was not markedly influenced by formula composition. Cells from mucoid and nonmucoid colonies formed by two strains on violet red bile glucose agar supplemented with pyruvate were inoculated into a milk-based powdered infant formula and a soybean-based powdered infant formula having a high aw range of 0.43 to 0.86 and stored at 4, 21, and 30 degrees C for up to 36 weeks. With few exceptions, populations of both strains decreased significantly in both formulas within 2 weeks at all temperatures; rates of death increased with increased aw and storage temperature. The presence of mucoidal extracellular materials on the surface of E. sakazakii cells was not associated with protection against death. This study shows that the retention of viability of E. sakazakii in powdered infant formula is affected by a, and temperature. Increases in both parameters cause an increase in the rate of death.

Atmospheric cold plasma inactivation of aerobic microorganisms on blueberries and effects on quality attributes

Cold plasma (CP) is a novel nonthermal technology, potentially useful in food processing settings. Berries were treated with atmospheric CP for 0, 15, 30, 45, 60, 90, or 120 s at a working distance of 7.5 cm with a mixture of 4 cubic feet/minute (cfm) of CP jet and 7 cfm of ambient air. Blueberries were sampled for total aerobic plate count (APC) and yeast/molds immediately after treatment and at 1, 2, and 7 days. Blueberries were also analyzed for compression firmness, surface color, and total anthocyanins immediately after each treatment. All treatments with CP significantly (P < 0.05) reduced APC after exposure, with reductions ranging from 0.8 to 1.6 log CFU/g and 1.5 to 2.0 log CFU/g compared to the control after 1 and 7 days, respectively. Treatments longer than 60s resulted in significant reductions in firmness, although it was demonstrated that collisions between the berries and the container contributed significantly to softening. A significant reduction in anthocyanins was observed after 90 s. The surface color measurements were significantly impacted after 120 s for the L* and a* values and 45 s for the b* values. CP can inactivate microorganisms on blueberries and could be optimized to improve the safety and quality of produce.

Survival and growth of Enterobacter sakazakii in infant rice cereal reconstituted with water, milk, liquid infant formula, or apple juice

Aims:  To determine survival and growth characteristics of Enterobacter sakazakii in infant rice cereal as affected by type of liquid used for reconstitution and storage temperature after reconstitution.

Inactivation of Listeria innocua, Salmonella Typhimurium, and Escherichia coli O157:H7 on surface and stem scar areas of tomatoes using in-package ozonation.

A novel in-package ozonation device was evaluated for its efficacy in inactivating three microorganisms (viz., Listeria innocua, attenuated Salmonella Typhimurium, and Escherichia coli O157:H7) on tomatoes and for its effect on fruit quality. The device produced ozone inside sealed film bags, reaching a concentration of 1,000 ppm within 1 min of activation. The three bacterial cultures were inoculated onto either the smooth surface or the stem scar areas of the tomatoes, which were then sealed in plastic film bags and subjected to in-package ozonation. L. innocua on tomatoes was reduced to nondetectable levels within 40 s of treatment on the tomato surface, with inactivation of ca. 4 log CFU per fruit on the stem scar area. An increase in treatment time did not result in a proportional increase in bacterial reduction. For E. coli O157:H7 and Salmonella, there was little difference (<1 log) in the effectiveness of the system when comparing surface and scar-inoculated bacteria. Both bacteria were typically reduced by 2 to 3 log CFU per fruit after 2- to 3-min treatments. No negative effects on fruit color or texture were observed during a 22-day posttreatment storage study of ozone-treated tomatoes. These results suggest that the three bacteria responded differently to ozonation and that in-package ozonation may provide an alternative to chemical sanitizers commonly used by the industry.

High-Pressure Inactivation of Human Norovirus Virus-Like Particles Provides Evidence that the Capsid of Human Norovirus Is Highly Pressure Resistant

ABSTRACT Human norovirus (NoV) is the leading cause of nonbacterial acute gastroenteritis epidemics worldwide. High-pressure processing (HPP) has been considered a promising nonthermal processing technology to inactivate food- and waterborne viral pathogens. Due to the lack of an effective cell culture method for human NoV, the effectiveness of HPP in inactivating human NoV remains poorly understood. In this study, we evaluated the effectiveness of HPP in disrupting the capsid of human NoV based on the structural and functional integrity of virus-like particles (VLPs) and histo-blood group antigen (HBGA) receptor binding assays. We found that pressurization at 500 to 600 MPa for 2 min, a pressure level that completely inactivates murine norovirus and feline calicivirus, was not sufficient to disrupt the structure and function of human NoV VLPs, even with a holding time of 60 min. Degradation of VLPs increased commensurate with increasing pressure levels more than increasing time. The times required for complete disruption of human NoV VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively. Human NoV VLPs were more resistant to HPP in their ability to bind type A than type B and O HBGAs. Additionally, the 23-nm VLPs appeared to be much more stable than the 38-nm VLPs. Taken together, our results demonstrated that the human NoV capsid is highly resistant to HPP. While human NoV VLPs may not be fully representative of viable human NoV, destruction of the VLP capsid is highly suggestive of a typical response for viable human NoV.