M. E. Berrang

Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere

The effects of controlled atmosphere storage (CAS) on survival and growth of Listeria monocytogenes on fresh asparagus, broccoli, and cauliflower were investigated. Vegetables were inoculated with L. monocytogenes strain Scott A or LCDC 81-861 (103-105 CFU/g) and stored at 4 and 15°C under CAS and air. L. monocytogenes populations were monitored over 21 day (4°C) and 10 day (15°C) periods using selective recovery media and a direct plating technique. CAS lengthened the time that all vegetables were considered acceptable for consumption by subjective inspection. Populations of L. monocytogenes increased during storage but CAS did not influence the rate of growth.

Broiler Carcass Contamination with Campylobacter from Feces during Defeathering

Three sets of experiments were conducted to explore the increase in recovery of Campylobacter from broiler carcasses after defeathering. In the first set of experiments, live broilers obtained from a commercial processor were transported to a pilot plant, and breast skin was sampled by a sponge wipe method before and after defeathering. One of 120 broiler breast skin samples was positive for Campylobacter before defeathering, and 95 of 120 were positive after defeathering. In the second set of experiments, Campylobacter-free flocks were identified, subjected to feed withdrawal, and transported to the pilot plant. Carcasses were intracloacally inoculated with Campylobacter (10(7) CFU) just prior to entering the scald tank. Breast skin sponge samples were negative for Campylobacter before carcasses entered the picker (0 of 120 samples). After defeathering, 69 of 120 samples were positive for Campylobacter, with an average of log10 2.7 CFU per sample (approximately 30 cm2). The third set of experiments was conducted using Campylobacter-positive broilers obtained at a commercial processing plant and transported live to the pilot plant. Just prior to scalding, the cloacae were plugged with tampons and sutured shut on half of the carcasses. Plugged carcasses were scalded, and breast skin samples taken before and after defeathering were compared with those collected from control broilers from the same flock. Prior to defeathering, 1 of 120 breast skin sponge samples were positive for the control carcasses, and 0 of 120 were positive for the plugged carcasses. After passing through the picker, 120 of 120 control carcasses had positive breast skin sponge samples, with an average of log10 4.2 CFU per sample (approximately 30 cm2). Only 13 of 120 plugged carcasses had detectable numbers of Campylobacter on the breast skin sponge, with an average of log10 2.5 CFU per sample. These data indicate that an increase in the recovery of Campylobacter after defeathering can be related to the escape of contaminated feces from the cloaca during defeathering.

Identification of a New Source of Campylobacter Contamination in Poultry: Transmission from Breeder Hens to Broiler Chickens

SUMMARY. Campylobacter jejuni, a foodborne pathogen closely associated with market poultry, is considered to be the most frequent agent of human gastroenteritis in the United States. The pathways involved in the contamination of poultry flocks, vertical transmission and/or horizontal transmission, are unclear. In this study, Campylobacter isolates from two independent commercial broiler breeder flocks, as well as from their respective progeny, were characterized and compared by PstI ribotype analysis and by DNA sequence analysis of the short variable region (SVR) of the flaA gene (flaA SVR). Campylobacter isolates originating from one set of breeder hens and the feces from their respective progeny demonstrated identical ribotype patterns as well as identical flaA SVR DNA sequences, thereby suggesting that these isolates were clonal in origin. Ribotype analysis of Campylobacter isolates from the second set of breeder hens and processed carcasses from their offspring resulted in two patterns. Sequence analysis placed these isolates into two closely related groups and one distant group, similar to the ribotype analysis. These results demonstrate that Campylobacter isolates from commercial broiler breeder flocks and from the respective broiler progeny may be of clonal origin and that breeder hens can serve as a source for Campylobacter contamination in poultry flocks.

Microbial, color and textural qualities of fresh asparagus, broccoli, and cauliflower stored under controlled atmosphere

Fresh asparagus, broccoli, and cauliflower were held under controlled atmosphere storage (CAS) and ambient air systems at 4°C for 21 days. Samples were examined on the initial day of experiments and after 2, 4, 7, 14, and 21 d of storage. On each day of analysis, color (value, chroma, and hue angle) was measured. Texture was measured as J needed to shear the vegetable/mm2 vegetable shear surface area. Total aerobic microbial populations were enumerated. Populations of total aerobic microorganisms were initially about 104 - 105 CFU/g on all three vegetables and grew to at least 107 CFU by d 21 of storage. CAS storage significantly reduced the growth of microorganisms on broccoli but had no significant effect with the other vegetables. Asparagus stored under CAS was easier to shear and had slightly higher hue angles than those of asparagus stored under air. Cauliflower stored under CAS showed less decline in value. CAS extended the length of time vegetables were subjectively considered acceptable for consumption. However, overall results indicate that quality of vegetables, as determined by objective measurements of color and texture, is not significantly influenced by CAS during refrigerated storage.

Novel Epidemic Clones of Listeria monocytogenes, United States, 2011

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Multilocus Sequence Typing of Listeria monocytogenes by Use of Hypervariable Genes Reveals Clonal and Recombination Histories of Three Lineages

ABSTRACT In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes, the sequences of all of the annotated genes from the fully sequenced strain L. monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L. monocytogenes ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes (hisJ, cbiE, truB, ribC, comEA, purM, aroE, hisC, and addB) in the 80 to 90% identity group and two genes (gyrB and rnhB) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L. monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of L. monocytogenes that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the L. monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of L. monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.

Effect of Intestinal Content Contamination on Broiler Carcass Campylobacter Counts

Intestinal contents may contaminate broiler carcasses during processing. The objective of this study was to determine what effect various levels of intestinal contents had on the numbers of Campylobacter detected in broiler carcass rinse samples. Eviscerated broiler carcasses were collected from the shackle line in a commercial processing plant immediately after passing through an inside/outside washer. Broiler carcasses were cut longitudinally into contralateral halves using a sanitized saw. Cecal contents from the same flock were collected, pooled, homogenized, and used to contaminate carcass halves. Paired carcass halves were divided into groups of eight each, and then cecal contents (2, 5, 10, 50, or 100 mg) were placed onto one randomly selected half of each carcass, while the corresponding half of the same broiler carcass received no cecal contents. Campylobacter counts from carcass halves with cecal contamination were compared to the uncontaminated halves of the same carcasses using a paired t test. Carcass halves with 5 mg or more of surface cecal contamination had significantly higher numbers of Campylobacter than those without (P < 0.01). Carcass halves contaminated with only 5 mg of cecal contents had an average of 3.3 log CFU Campylobacter per ml of rinse, while corresponding uncontaminated carcass halves had 2.6 log CFU Campylobacter per ml of rinse. These data indicate that even small (5 mg) amounts of cecal contents can cause a significant increase in the numbers of Campylobacter on eviscerated broiler carcasses. Therefore, it is important to keep such contamination to a minimum during processing.

Direct microscopic observation and viability determination of Campylobacter jejuni on chicken skin.

A method was developed to determine the survival of Campylobacter jejuni at specific sites on chicken skin, and this method was used to observe the survival of C. jejuni at various locations on the skin during storage. This method uses confocal scanning laser microscopy to visualize C. jejuni transformed with P(c)gfp plasmid (GFP-Campylobacter) and stained with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). The green fluorescence of dead C. jejuni cells and the red fluorescent CTC-formazan in viable Campylobacter cells were clearly visible on chicken skin. The GFP-Campylobacter remaining on the chicken skin surface after rinsing was mostly located in crevices, entrapped inside feather follicles with water, and entrapped in the surface water layer. Most viable cells were entrapped with water in the skin crevices and feather follicles. These sites provide a suitable microenvironment for GFP-Campylobacter to survive. The population of C. jejuni on chicken skin decreased by 1 log unit during storage at 25 degrees C for 24 h. C. jejuni located in sites 20 to 30 microm beneath the chicken skin surface maintained viability during incubation at 25 degrees C. C. jejuni on chicken skin stored at 4 degrees C maintained constant numbers during a 72-h incubation with no significant changes in population feather follicles or crevices. Live and dead cells were initially retained with water on the skin and penetrated into the skin follicles and channels during storage. Microscopic observations of GFP-producing cells allowed the identification of survival niches for C. jejuni present on chicken skin.

Cecal Carriage of Clostridium perfringens in Broiler Chickens Given Mucosal Starter Culture

Day-of-hatch broiler chicks housed in isolation units were each given, by oral gavage, 0.1 ml of Mucosal Starter Culture (MSC) or saline control. Each of the treated and control chicks was subsequently given a composite culture of three strains of bacitracin-resistant Clostridium perfringens (Cp) previously isolated from chickens with symptoms of necrotic enteritis. Some chicks were maintained on a corn-based diet provided ad libitum. Others were given the feed supplemented with 50% rye (a predisposing factor for necrotic enteritis). At 7, 14, and 21 days after receiving Cp, chicks were euthanatized, and cecal contents were diluted and plated on selective agar containing bacitracin. For chicks on corn feed, Cp numbers were similar in control birds and birds given MSC in three of four trials. In two of the trials that demonstrated no effect of MSC on Cp numbers, enterotoxin presence was determined. The number of birds with detectable Cp enterotoxin in their small intestine and the mean toxin levels were lower in the MSC-treated birds. In a fourth trial with birds on corn-based feed, mean Cp numbers and the number of Cp-positive birds were lower in the MSC-treated birds. For the two trials involving chickens on rye-supplemented feed, Cp numbers and the percentage of Cp-positive birds were significantly reduced in MSC-treated birds compared with control birds. Enterotoxin in birds receiving the 50% rye diet was at low levels or not detected in control and MSC-treated birds. Results suggest that MSC may reduce intestinal proliferation of Cp, a causative agent of necrotic enteritis in poultry and of foodborne disease in humans.

Molecular Characterization of Listeria monocytogenes Isolated from a Poultry Further Processing Facility and from Fully Cooked Product

This study was undertaken to explore environmental sources of Listeria monocytogenes in a commercial chicken further processing facility and to compare the isolates obtained from this facility with others obtained from fully cooked product. In a survey conducted at the processing facility, 40 environmental sites (encompassing two production lines and representing areas in which raw and cooked products are processed) were cultured for L. monocytogenes. The resulting isolates were subjected to molecular subtyping by ribotyping, and these isolates were compared with 25 isolates collected by plant personnel from product contact surfaces and from fully cooked product. Eighty-nine environmental and product isolates were divided into 15 distinct ribogroups. Two ribogroups included isolates from fully cooked product; the members of these two ribogroups were subjected to further analysis by pulsed-field gel electrophoresis, resulting in four clusters. L. monocytogenes isolates from fully cooked product produced on line 1 were found to be indistinguishable from isolates collected from (i) drains on the raw-product side of line 1 and (ii) the floor surface in the cooked-product area of line 1. L. monocytogenes isolates from fully cooked product from line 2 were found to be indistinguishable from isolates collected from (i) the spiral freezer exit conveyor on line 2, (ii) raw product contact surfaces on line 1, and (iii) drains in the cooked-product area of line 1. These data suggest that L. monocytogenes can colonize a poultry further processing facility and eventually be transferred to fully cooked product.