Jeffrey M. Farber

Changes in populations of Listeria monocytogenes inoculated on packaged fresh-cut vegetables

A variety of wholesale and retail packaged vegetables and salads were inoculated with a mixture of strains of Listeria monocytogenes and incubated at 4 and 10 degrees C. Whole rutabagas, butternut squash, and onions, as well as packaged Caesar salad, carrots, coleslaw mix, and stir-fry vegetables were purchased from local supermarkets in the Ottawa area. L. monocytogenes population levels remained constant on all fresh-cut vegetables stored at 4 degrees C for 9 days, except for carrots and butternut squash: counts of cell numbers declined on carrots and increased on the butternut squash. Fresh-cut vegetables stored at 10 degrees C, however, supported good growth of L. monocytogenes on all vegetables tested, except for chopped carrots, where the population decreased approximately 2 log units over a 9-day storage period. As in the situation with the produce stored at 4 degrees C, butternut squash supported the highest rate of cell growth. In addition, Caesar salad and coleslaw mix were kept at 25 degrees C for 1 or 2 days before subsequent storage at 4 or 10 degrees C to stimulate extreme temperature-abuse conditions. In Caesar salad stored at 4 degrees C, by day 6 an initial 24- and 48-h temperature abuse at 25 degrees C led to a 1.21- and 2.55-log-unit population increase, respectively, over the control. Similar increases were observed on Caesar salads stored at 10 degrees C. Compared to Caesar salad, coleslaw mix temperature-abused at 25 degrees C and then stored at 4 degrees C supported slightly greater increases in the population of L. monocytogenes, i.e., a 3.22- and 3.83-log-unit increase over the control for the 1- and 2-day abused samples, respectively. Coleslaw mix samples temperature-abused and then stored at 10 degrees, however, only showed log unit increases of 1.75 and 1.94, respectively, compared to the controls. These results point to the importance of strict temperature control to prevent or reduce the growth of L. monocytogenes cells on fresh-cut vegetables.

Enterobacter sakazakii: a review

Enterobacter sakazakii, previously referred to as a yellow-pigmented Enterobacter cloacae was designated as a unique species in 1980. This reclassification was based on differences from E. cloacae in DNA relatedness, pigment production and biochemical reactions. E. sakazakii has been implicated in a severe form of neonatal meningitis. Although studies have failed to identify an environmental source for the organism, dried-infant formula has been implicated in both outbreaks and sporadic cases of E. sakazakii meningitis. The high mortality rate (40-80%), the severity of the infection in infants, plus the scarcity of information on the ecology and pathogenicity of this organism warranted a review of the clinical and microbiological features of this putative foodborne pathogen.

Incidence, survival, and growth of Enterobacter sakazakii in infant formula

Enterobacter sakazakii has been implicated in a severe form of neonatal meningitis. Although studies have failed to identify an environmental source for the organism, dried infant formula has been implicated in outbreaks and sporadic cases of E. sakazakii meningitis. The high mortality rate (50 to 75%), the severity of the infection in infants, and the lack of information on the incidence, survival, and growth of E. sakazakii in foods led to this study. Experiments were undertaken to determine the incidence of E. sakazakii in dried infant formula, the temperature range for growth, and the growth characteristics of E. sakazakii in reconstituted dried infant formula. Strains of E. sakazakii were isolated from dried infant formula available on the Canadian retail market. The prevalence varied from 0 to 12% in samples from five different companies. For both clinical and food isolates, minimum growth temperatures of 5.5 to 8.0°C were observed by using a temperature-gradient incubator. The potential growth of E. sakazakii was followed by using a mixture of food and clinical isolates in three different formulas incubated at 4, 10, and 23°C. Average generation times were 40 min at 23°C and 4.98 h at 10°C. E. sakazakii strains did not grow at 4°C and began to die off during storage at this temperature. The results of this study stress the importance of using aseptic methods and proper temperature control in the preparation, use, and storage of dried infant formula.

Contamination of foods by food handlers: experiments on hepatitis A virus transfer to food and its interruption.

ABSTRACT Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm2resulted in transfer of 9.2% ± 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% ± 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.

Health risk assessment of Listeria monocytogenes in Canada

In this review, the major steps used in the formulation of a health risk assessment for Listeria monocytogenes in foods are discussed. Data is given on the numbers of human listeriosis cases reported in Canada along with the current Canadian regulatory policy on L. monocytogenes. Four major steps in the health risk assessment of this organism in foods, namely, hazard identification, hazard characterization, exposure assessment and risk characterization, were examined. For hazard characterization, since it is known that no direct human dose response data is available for L.monocytogenes, a flexible dose response model called the Weibull-Gamma model was evaluated. For the exposure assessment, pâté and soft cheese, both high-risk foods in terms of listeriosis infection, were used as prototypes in some of the models that were used. Using disappearance data for cheese and 100 g as a typical serving, the data suggested an average of 102 servings per capita, per year in Canada. As a rough approximation, for L. monocytogenes, reference ID10 and ID90 dose levels of response for both normal and high risk populations were given as 10(7) and 10(9) for normal individuals, and 10(5) and 10(7) for high-risk people. The corresponding dose response models were graphically displayed. These models exhibited a higher degree of susceptibility and less host/pathogen heterogeneity for the higher risk group. The range of doses between the ID10 and ID90 reference values corresponded roughly to levels associated with cases of listeriosis. In the risk characterization stage, dose response data was combined with some predictive growth modeling data of L. monocytogenes on pâté, assuming an initial exposure of a single cell for food stored at 4 degrees and 8 degrees C. Storage of pâté at 4 degrees C for more than 35 days resulted in a rapidly increasing risk for the high risk population, while storage at 8 degrees C produced a similar risk after about 13 days. In addition, an equation, used to calculate the average probability of acquiring human listeriosis in Canada from soft and semi-soft cheese consumption, was formulated. Computations derived from this equation indicated a substantial level consistency between reported data and assumptions of the risk assessment model. An important part of risk characterization or possibly risk management is characterizing the economic and social consequences of estimated risks. The total annual estimated cost of listeriosis illnesses and deaths in Canada was estimated to be between 11.1 and 12.6 million dollars.

Human noroviruses in swine and cattle.

Detection of GII.4 norovirus sequences in animal fecal samples and retail meats demonstrates that noroviruses may be transmitted zoonotically.

Enterobacter sakazakii: Infectivity and Enterotoxin Production In Vitro and In Vivo

Enterobacter sakazakii has been implicated as the causal organism in a severe form of neonatal meningitis, with reported mortality rates of 40 to 80%. Dried infant formula has been identified as a potential source of the organism in both outbreaks and sporadic cases. In this study, clinical and foodborne isolates of E. sakazakii were evaluated for enterotoxin production by the suckling mouse assay. In addition, suckling mice were challenged both orally and by intraperitoneal injection. Of 18 E. sakazakii strains evaluated, four were found to test positive for enterotoxin production. All strains of E. sakazakii were lethal to suckling mice at 10(8) CFU per mouse by intraperitoneal injection, while two strains caused death by the peroral route. In in vitro assays, CHO, Vero, and Y-1 cells demonstrated both cell lysis and rounding when exposed to E. sakazakii strain LA filtrates. This is the first report describing any putative virulence factors of E. sakazakii.

Standardization of a Method To Determine the Efficacy of Sanitizers in Inactivating Human Pathogenic Microorganisms on Raw Fruits and Vegetables

The efficacy of sanitizers in killing human pathogenic microorganisms on a wide range of whole and fresh-cut fruits and vegetables has been studied extensively. Numerous challenge studies to determine the effects of storage conditions on survival and growth of pathogens on raw produce have also been reported. Results of these studies are often difficult to assess because of the lack of sufficient reporting of methods or, comparatively, because of variations in procedures for preparing and applying inocula to produce, conditions for treatment and storage, and procedures for enumerating pathogens. There is a need for a standard method to accurately determine the presence and populations of pathogenic microorganisms on produce. The adoption of standard, well-characterized reference strains would benefit a comparative assessment of a basic method among laboratories. A single protocol will not be suitable for all fruits and vegetables. Modifications of a basic method will be necessary to achieve maximum recovery of pathogens on various types of produce subjected to different sanitizer or storage treatments. This article discusses parameters that must be considered in the course of developing a basic standard method against which these modifications could be made.

Large-Scale Comparative Genomics Meta-Analysis of Campylobacter jejuni Isolates Reveals Low Level of Genome Plasticity

ABSTRACT We have used comparative genomic hybridization (CGH) on a full-genome Campylobacter jejuni microarray to examine genome-wide gene conservation patterns among 51 strains isolated from food and clinical sources. These data have been integrated with data from three previous C. jejuni CGH studies to perform a meta-analysis that included 97 strains from the four separate data sets. Although many genes were found to be divergent across multiple strains (n = 350), many genes (n = 249) were uniquely variable in single strains. Thus, the strains in each data set comprise strains with a unique genetic diversity not found in the strains in the other data sets. Despite the large increase in the collective number of variable C. jejuni genes (n = 599) found in the meta-analysis data set, nearly half of these (n = 276) mapped to previously defined variable loci, and it therefore appears that large regions of the C. jejuni genome are genetically stable. A detailed analysis of the microarray data revealed that divergent genes could be differentiated on the basis of the amplitudes of their differential microarray signals. Of 599 variable genes, 122 could be classified as highly divergent on the basis of CGH data. Nearly all highly divergent genes (117 of 122) had divergent neighbors and showed high levels of intraspecies variability. The approach outlined here has enabled us to distinguish global trends of gene conservation in C. jejuni and has enabled us to define this group of genes as a robust set of variable markers that can become the cornerstone of a new generation of genotyping methods that use genome-wide C. jejuni gene variability data.

Norovirus Cross-Contamination during Food Handling and Interruption of Virus Transfer by Hand Antisepsis: Experiments with Feline Calicivirus as a Surrogate †

While there is good epidemiological evidence for foods as vehicles for norovirus transmission, the precise means of spread and its control remain unknown. The feline calicivirus was used as a surrogate for noroviruses to study infectious virus transfer between hands and selected types of foods and environmental surfaces. Assessment of the potential of selected topicals in interrupting such virus transfer was also made. Ten microliters of inoculum of feline calicivirus deposited onto each fingerpad of adult subjects was allowed to air dry and the contaminated area on individual fingerpads was pressed (10 s at a pressure of 0.2 to 0.4 kg/cm2) onto 1-cm-diameter disks of ham, lettuce, or brushed stainless steel. The virus remaining on the donor and that transferred to the recipient surfaces was eluted and plaque assayed. Virus transfer to clean hands from experimentally contaminated disks of ham, lettuce, and stainless steel was also tested. Nearly 46 +/- 20.3, 18 +/- 5.7, and 13 +/- 3.6% of infectious virus was transferred from contaminated fingerpads to ham, lettuce, and metal disks, respectively. In contrast, approximately 6 +/- 1.8, 14 +/- 3.5, and 7 +/- 1.9% virus transfer occurred, respectively, from ham, lettuce, and metal disks to hands. One-way analysis of variance test showed that pretreatment (washing) of the fingerpads either with water or with both topical agent and water significantly (P < 0.05) reduced virus transfer to < or = 0.9%, as compared with < or = 2.3 and < or = 3.4% transfer following treatments with either 75% (vol/vol) ethanol or a commercial hand gel containing 62% ethanol, respectively. Despite wide variations in virus transfer among the targeted items used, intervention agents tested reduced virus transfer significantly (P < 0.05) when compared with that without such treatments (71 +/- 8.9%). These findings should help in a better assessment of the potential for cross-contamination of foods during handling and also assist in developing more effective approaches to foodborne spread of norovirus infections.