Population structure of Listeria monocytogenes serotype 4b isolates from sporadic human listeriosis cases in the United States from 2003 to 2008.

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis.

Listeria monocytogenes in donated platelets: a potential transfusion-transmitted pathogen intercepted through screening.

BACKGROUND: Bacterial contamination of blood components is a potentially life-threatening complication of transfusions. In October 2005, the Centers for Disease Control and Prevention (CDC) noted four Listeria monocytogenes (Lm) isolates cultured from four different units of donated apheresis platelets (PLTs) among Lm isolates sent to the CDC National Listeria Reference Laboratory for subtyping as part of routine surveillance activities. STUDY DESIGN AND METHODS: We describe an investigation to determine possible common sources of infection among donors or factors associated with PLT collection or storage and to determine whether human transfusion-associated listeriosis cases had been reported. We also reviewed all isolates with PLTs as a source sent to the CDC National Listeria Reference Laboratory between November 1, 2005, and December 31, 2011. RESULTS: Each PLT donor-associated isolate had a distinct pulsed-field gel electrophoresis pattern combination. Other than these four cases, no other cases of Lm-contaminated PLTs were identified by the American Red Cross or by CDC during 2005. However, two additional cases of Lm isolated from donated PLTs were detected, one in 2008 and one in 2011. CONCLUSION: Although the source of contamination for these PLT units is unclear, and a source common to all units was not identified, this investigation underscores the value of screening for bacterial contaminants of PLTs.

Listeriosis outbreaks and associated food vehicles, United States, 1998-2008.

Listeria monocytogenes, a bacterial foodborne pathogen, can cause meningitis, bacteremia, and complications during pregnancy. This report summarizes listeriosis outbreaks reported to the Foodborne Disease Outbreak Surveillance System of the Centers for Disease Control and Prevention during 1998-2008. The study period includes the advent of PulseNet (a national molecular subtyping network for outbreak detection) in 1998 and the Listeria Initiative (enhanced surveillance for outbreak investigation) in 2004. Twenty-four confirmed listeriosis outbreaks were reported during 1998-2008, resulting in 359 illnesses, 215 hospitalizations, and 38 deaths. Outbreaks earlier in the study period were generally larger and longer. Serotype 4b caused the largest number of outbreaks and outbreak-associated cases. Ready-to-eat meats caused more early outbreaks, and novel vehicles (i.e., sprouts, taco/nacho salad) were associated with outbreaks later in the study period. These changes may reflect the effect of PulseNet and the Listeria Initiative and regulatory initiatives designed to prevent contamination in ready-to-eat meat and poultry products.

Invasive listeriosis in the Foodborne Diseases Active Surveillance Network (FoodNet), 2004-2009: further targeted prevention needed for higher-risk groups.

BACKGROUND: Listeriosis can cause severe disease, especially in fetuses, neonates, older adults, and persons with certain immunocompromising and chronic conditions. We summarize US population-based surveillance data for invasive listeriosis from 2004 through 2009. METHODS: We analyzed Foodborne Diseases Active Surveillance Network (FoodNet) data for patients with Listeria monocytogenes isolated from normally sterile sites. We describe the epidemiology of listeriosis, estimate overall and specific incidence rates, and compare pregnancy-associated and nonpregnancy-associated listeriosis by age and ethnicity. RESULTS: A total of 762 listeriosis cases were identified during the 6-year reporting period, including 126 pregnancy-associated cases (17%), 234 nonpregnancy-associated cases(31%) in patients aged <65 years, and 400 nonpregnancy-associated cases (53%) in patients aged ≥ 65 years. Eighteen percent of all cases were fatal. Meningitis was diagnosed in 44% of neonates. For 2004-2009, the overall annual incidence of listeriosis varied from 0.25 to 0.32 cases per 100,000 population. Among Hispanic women, the crude incidence of pregnancy-associated listeriosis increased from 5.09 to 12.37 cases per 100,000 for the periods of 2004-2006 and 2007-2009, respectively; among non-Hispanic women, pregnancy-associated listeriosis increased from 1.74 to 2.80 cases per 100,000 for the same periods. Incidence rates of nonpregnancy-associated listeriosis in patients aged ≥ 65 years were 4-5 times greater than overall rates annually. CONCLUSIONS: Overall listeriosis incidence did not change significantly from 2004 through 2009. Further targeted prevention is needed, including food safety education and messaging (eg, avoiding Mexican-style cheese during pregnancy). Effective prevention among pregnant women, especially Hispanics, and older adults would substantially affect overall rates.

Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette.

Listeria monocytogenes is the etiological agent of listeriosis, a severe food-borne illness. The population of L. monocytogenes is divided into four lineages (I to IV), and serotype 4b in lineage I has been involved in numerous outbreaks. Several serotype 4b epidemic-associated clonal groups (ECI, -II, and -Ia) have been identified. In this study, we characterized a panel of strains of serotype 4b that produced atypical results with a serotype-specific multiplex PCR and possessed the lmo0734 to lmo0739 gene cassette that had been thought to be specific to lineage II. The cassette was harbored in a genomically syntenic locus in these isolates and in lineage II strains. Three distinct clonal groups (groups 1 to 3) were identified among these isolates based on single-nucleotide polymorphism-based multilocus genotyping (MLGT) and DNA hybridization data. Groups 1 and 2 had MLGT haplotypes previously encountered among clinical isolates and were composed of clinical isolates from multiple states in the United States. In contrast, group 3 consisted of clinical and environmental isolates solely from North Carolina and exhibited a novel haplotype. In addition, all group 3 isolates had DNA that was resistant to MboI, suggesting methylation of adenines at GATC sites. Sequence analysis of the lmo0734 to lmo0739 gene cassette from two strains (group 1 and group 3) revealed that the genes were highly conserved (>99% identity). The data suggest relatively recent horizontal gene transfer from lineage II L. monocytogenes into L. monocytogenes serotype 4b and subsequent dissemination among at least three distinct clonal groups of L. monocytogenes serotype 4b, one of which exhibits restrictions in regional distribution.

Re-evaluation, optimization, and multilaboratory validation of the PulseNet-standardized pulsed-field gel electrophoresis protocol for Listeria monocytogenes.

The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest.

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.

Short-term genome evolution of Listeria monocytogenes in a non-controlled environment.

BACKGROUND: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses. RESULTS: The two L. monocytogenes isolates from 1988 and the two isolates from 2000 had highly similar genome backbone sequences with very few single nucleotide (nt) polymorphisms (1 - 8 SNPs/isolate; confirmed by re-sequencing). While no genome rearrangements were identified in the backbone genome of the four isolates, a 42 kb prophage inserted in the chromosomal comK gene showed evidence for major genome rearrangements. The human-food isolate pair from each 1988 and 2000 had identical prophage sequence; however, there were significant differences in the prophage sequences between the 1988 and 2000 isolates. Diversification of this prophage appears to have been caused by multiple homologous recombination events or possibly prophage replacement. In addition, only the 2000 human isolate contained a plasmid, suggesting plasmid loss or acquisition events. Surprisingly, besides the polymorphisms found in the comK prophage, a single SNP in the tRNA Thr-4 prophage represents the only SNP that differentiates the 1988 isolates from the 2000 isolates. CONCLUSION: Our data support the hypothesis that the 2000 human listeriosis outbreak was caused by a L. monocytogenes strain that persisted in a food processing facility over 12 years and show that genome sequencing is a valuable and feasible tool for retrospective epidemiological analyses. Short-term evolution of L. monocytogenes in non-controlled environments appears to involve limited diversification beyond plasmid gain or loss and prophage diversification, highlighting the importance of phages in bacterial evolution.

Listeria monocytogenes infection from foods prepared in a commercial establishment: a case-control study of potential sources of sporadic illness in the United States.

BACKGROUND: Listeria monocytogenes has been estimated to cause >2500 illnesses and 500 deaths annually in the United States. Efforts to reduce foodborne listeriosis have focused on foods frequently implicated in outbreaks. Potential sources for L. monocytogenes infection not associated with outbreaks remain poorly understood. METHODS: The Foodborne Diseases Active Surveillance Network conducts surveillance for culture-confirmed listeriosis at clinical laboratories in 9 states. After excluding outbreak-associated cases, we attempted to enroll eligible case patients with L. monocytogenes infection in a case-control study from 2000 through 2003. Control subjects were recruited through health care providers and were matched to case patients by state, age, and immunosuppression status. Data were collected about exposures occurring in the 4 weeks before specimen collection from the case patients. RESULTS: Of the 249 case patients with L. monocytogenes infection, only 12 (5%) had cases that were associated with outbreaks; 6 other patients were ineligible for other reasons. Of 231 eligible case patients, 169 (73%) were enrolled in the study. We classified 28 case patients as having pregnancy-associated cases. We enrolled 376 control subjects. In multivariable analysis, L. monocytogenes infection was associated with eating melons at a commercial establishment (odds ratio, 2.6; 95% confidence interval, 1.4-5.0) and eating hummus prepared in a commercial establishment (odds ratio, 5.7; 95% confidence interval, 1.7-19.1). CONCLUSIONS: Most cases of L. monocytogenes infection were not associated with outbreaks. Reducing the burden of foodborne listeriosis may require interventions directed at retail environments and at foods, such as melons and hummus, that are not commonly recognized as high risk. Because of the severity of listeriosis, pregnant women and other persons at risk may wish to avoid eating these newly implicated foods.

Reduction in the incidence of invasive listeriosis in foodborne diseases active surveillance network sites, 1996-2003.

BACKGROUND: Listeriosis is a leading cause of death among patients with foodborne diseases in the United States. Monitoring disease incidence is an important element of listeriosis surveillance and control. METHOD: We conducted population-based surveillance for Listeria monocytogenes isolates obtained from normally sterile sites at all clinical diagnostic laboratories in the Foodborne Diseases Active Surveillance Network from 1996 through 2003. RESULTS: The incidence of laboratory-confirmed invasive listeriosis decreased by 24% from 1996 through 2003; pregnancy-associated disease decreased by 37%, compared with a decrease of 23% for patients > or =50 years old. The highest incidence was reported among Hispanic persons from 1997 through 2001. Differences in incidence by age group and ethnicity may be explained by dietary preferences. CONCLUSION: The marked decrease in the incidence of listeriosis may be related to the decrease in the prevalence of L. monocytogenes contamination of ready-to-eat foods since 1996. The crude incidence in 2003 of 3.1 cases per 1 million population approaches the government's Healthy People objective of 2.5 cases per 1 million population by 2005. Further decreases in listeriosis incidence will require continued efforts of industry and government to reduce contamination of food and continued efforts to educate consumers and clinicians.

Multistate outbreak of Listeriosis linked to turkey deli meat and subsequent changes in US regulatory policy.

BACKGROUND: Listeriosis, a life-threatening foodborne illness caused by Listeria monocytogenes, affects approximately 2500 Americans annually. Between July and October 2002, an uncommon strain of L. monocytogenes caused an outbreak of listeriosis in 9 states. METHODS: We conducted case finding, a case-control study, and traceback and microbiological investigations to determine the extent and source of the outbreak and to propose control measures. Case patients were infected with the outbreak strain of L. monocytogenes between July and November 2002 in 9 states, and control patients were infected with different L. monocytogenes strains. Outcome measures included food exposure associated with outbreak strain infection and source of the implicated food. RESULTS: Fifty-four case patients were identified; 8 died, and 3 pregnant women had fetal deaths. The case-control study included 38 case patients and 53 control patients. Case patients consumed turkey deli meat much more frequently than did control patients (P = .008, by Wilcoxon rank-sum test). In the 4 weeks before illness, 55% of case patients had eaten deli turkey breast more than 1-2 times, compared with 28% of control patients (odds ratio, 4.5; 95% confidence interval, 1.3-17.1). Investigation of turkey deli meat eaten by case patients led to several turkey processing plants. The outbreak strain was found in the environment of 1 processing plant and in turkey products from a second. Together, the processing plants recalled > 30 million pounds of products. Following the outbreak, the US Department of Agriculture's Food Safety and Inspection Service issued new regulations outlining a L. monocytogenes control and testing program for ready-to-eat meat and poultry processing plants. CONCLUSIONS: Turkey deli meat was the source of a large multistate outbreak of listeriosis. Investigation of this outbreak helped guide policy changes designed to prevent future L. monocytogenes contamination of ready-to-eat meat and poultry products.

Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard.

The performance of a multiplex PCR assay that separates the four major serovars of the pathogenic Listeria monocytogenes into four distinct PCR groups was evaluated through a multicenter typing study. Identical panels of 90 Listeria isolates were distributed to five participating laboratories that were blind to the nature of the isolates. Isolates were characterized using the previously standardized protocol. Overall concordance was 96.6 to 100%, sufficient for the assay to be used as an alternative to serotyping and confidently applied in laboratories involved in L. monocytogenes typing.

Microbiological aspects of the investigation that traced the 1998 outbreak of listeriosis in the United States to contaminated hot dogs and establishment of molecular subtyping-based surveillance for Listeria monocytogenes in the PulseNet network.

A multistate outbreak of listeriosis occurred in the United States in 1998 with illness onset dates between August and December. The outbreak caused illness in 108 persons residing in 24 states and caused 14 deaths and four miscarriages or stillbirths. This outbreak was detected by public health officials in Tennessee and New York who observed significant increases over expected listeriosis cases in their states. Subsequently, the Centers for Disease Control and Prevention (CDC) began laboratory characterization of clinical isolates of Listeria monocytogenes by serotyping and restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis (PFGE). For the purpose of this investigation, outbreak-related isolates were defined as those that had a specific AscI-PFGE pattern and indistinguishable or highly similar (no more than 2 band difference in 26 bands) ApaI-PFGE patterns when their DNA was restricted by AscI and ApaI restriction enzymes. Timely availability of molecular subtyping results enabled epidemiologists to separate outbreak cases from temporally associated sporadic cases in the same geographic areas and facilitated the identification of contaminated hot dogs manufactured at a single commercial facility as the source of the outbreak. During the investigation of this outbreak, a standardized protocol for subtyping L. monocytogenes by PFGE was developed and disseminated to public health laboratories participating with CDC's PulseNet network; these laboratories were requested to begin routine PFGE subtyping of L. monocytogenes.

Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat.

BACKGROUND: Despite a decreasing incidence of listeriosis in the United States, molecular subtyping has increased the number of recognized outbreaks. In September 2000, the New York City Department of Health identified a cluster of infections caused by Listeria monocytogenes isolates with identical molecular subtypes by pulsed-field gel electrophoresis (PFGE) and ribotyping. METHODS: To determine the magnitude of the outbreak and identify risk factors for infection, we notified state health departments and conducted a case-control study. A case was defined as a patient or mother-infant pair infected with Listeria monocytogenes whose isolate yielded the outbreak PFGE pattern. Controls were patients infected with Listeria monocytogenes whose isolate yielded a different PFGE pattern. Patients were asked about food and drink consumed during the 30 days before the onset of illness. RESULTS: Between May and December 2000, there were 30 clinical isolates of Listeria monocytogenes with identical PFGE patterns identified in 11 US states. Cases of infection caused by these isolates were associated with 4 deaths and 3 miscarriages. A case-control study implicated sliced processed turkey from a delicatessen (Mantel-Haenszel odds ratio, 8.0; 95% confidence interval, 1.2-43.3). A traceback investigation identified a single processing plant as the likely source of the outbreak, and the company voluntarily recalled 16 million pounds of processed meat. The same plant had been identified in a Listeria contamination event that had occurred more than a decade previously. CONCLUSIONS: Prevention of persistent L. monocytogenes contamination in food processing plants presents a critical challenge to food safety professionals.

Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard.

The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described.

Outbreak of listeriosis among Mexican immigrants as a result of consumption of illicitly produced Mexican-style cheese.

BACKGROUND: In 2000, an outbreak of listeriosis among Hispanic persons was identified in Winston-Salem, North Carolina. The objectives of the present study were to identify the source of, strains associated with, and risk factors for Listeria monocytogenes infection for patients affected by the outbreak. METHODS: Microbiological, case-control, and environmental investigations were conducted. Participants in the case-control study were case patients who became infected with L. monocytogenes between 1 October 2000 and 31 January 2001 and control subjects who were matched with case patients on the basis of ethnicity, sex, age, and pregnancy status. All participants were residents of Winston-Salem. RESULTS: We identified 13 patients, all of whom were Hispanic, including 12 females who were 18-38 years of age. Eleven case patients were pregnant; infection with L. monocytogenes resulted in 5 stillbirths, 3 premature deliveries, and 3 infected newborns. Case patients were more likely than control subjects to have eaten the following foods: fresh, unlabeled, Mexican-style cheese sold by door-to-door vendors (matched odds ratio [MOR], 17.5; 95% confidence interval [CI], 2.0-152.5); queso fresco, a Mexican-style soft cheese (MOR, 7.3; 95% CI, 1.4-37.5); and hot dogs (MOR, 4.6; 95% CI, 1.1-19.4). L. monocytogenes isolates recovered from 10 female case patients, from cheese bought from a door-to-door vendor, from unlabeled cheese from 2 Hispanic markets, and from raw milk from a local dairy had indistinguishable patterns on pulsed-field gel electrophoresis. CONCLUSIONS: This outbreak of listeriosis was caused by noncommercial, fresh, Mexican-style cheese made from contaminated raw milk traced to 1 local dairy. We recommend educating Hispanic women about food safety while they are pregnant, enforcing laws that regulate the sale of raw milk and dairy products made by unlicensed manufacturers, making listeriosis a reportable disease in all states, routinely interviewing case patients, and routinely subtyping clinical L. monocytogenes isolates.

Genetic markers unique to Listeria monocytogenes serotype 4b differentiate epidemic clone II (hot dog outbreak strains) from other lineages.

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.

Comparison of Three Selective Enrichment Methods for the Isolation of Listeria monocytogenes from Naturally Contaminated Foods.

Three selective enrichment procedures-the U.S. Food and Drug Administration (FDA) method, the U.S. Department of Agriculture (USDA) method, and the Netherlands Government Food Inspection Service (NGFIS) method-were compared for isolating Listeria monocytogenes from contaminated foods. The foods were obtained from the refrigerators of patients with culture-proven listeriosis who were identified through multistate active surveillance in a U.S. population of 19 million. The study was designed to identify foods that may be important in transmission of L. monocytogenes in sporadic cases of human listeriosis. Of 899 foods analyzed by all three methods, 121 were positive for L. monocytogenes by at least one method. The three enrichment methods detected L. monocytogenes in 65% (FDA), 74% (USDA), and 74% (NGFIS) of the foods shown to contain L. monocytogenes . The differences among the three methods were not statistically significant. However, the recovery of L. monocytogenes by a combination of any two methods (USDA-FDA 88%, USDA-NGFIS 91%, FDA-NGFIS 87%) was significantly better than that by one method alone (p < 0.02). The differences among the combinations of methods were not statistically significant. These results suggest that at least two enrichment methods must be used in combination to recover L. monocytogenes from contaminated foods with a success rate near 90%. Correlations were observed between negative results and low (<0.3 CFU/g) level of L. monocytogenes contamination for the USDA (p << 0.001) and NGFIS (p << 0.001) methods. A similar but somewhat weaker association was observed for the FDA method (p < 0.06).