Search

Background: Molecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST)., Methods: Our study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak., Results: Out of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient's refrigerator., Conclusions: Our findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009-2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

A longitudinal study was conducted to determine the prevalence of Listeria spp. in a commercial fresh mushroom slicing and packaging environment. Samples were collected at three different sampling periods within a 13-month time interval. Of the 255 environmental samples collected, 18.8% tested positive for L. monocytogenes, 4.3% for L. innocua, and 2.0% for L. grayi. L. monocytogenes was most often found on wet floors within the washing and slicing and packaging areas. Each of the 171 L. monocytogenes isolates found in the environment could be placed into one of three different serotypes; 1/2c was predominant (93.6%), followed by 1/2b (3.5%) and 1/2a (2.9%). Of 58 isolates subtyped using multi-virulence-locus sequence typing, all 1/2c isolates were identified as virulence type (VT) 11 (VT11), all 1/2b isolates were VT105, and 1/2a isolates were either VT107 or VT56. VT11 was designated as the predominant and persistent clone in the environment because it was isolated repeatedly at numerous locations throughout the study. The overall predominance and persistence of VT11 indicates that it likely colonized the mushroom processing environment. Areas adjacent to the trench drain in the washing and slicing area and a floor crack in the packaging area may represent primary harborage sites (reservoirs) for VT11. Improvements made to sanitation procedures by company management after period 2 coincided with a significant (P ≤ 0.001) reduction in the prevalence of L. monocytogenes from 17.8% in period 1 and 30.7% in period 2 to 8.5% in period 3. This suggests that targeted cleaning and sanitizing procedures can be effective in minimizing the occurrence of L. monocytogenes contamination in processing facilities. Additional research is needed to understand why VT11 was predominant and persistent in the mushroom processing environment.

A two-temperature agar diffusion bioassay is commonly used to quantify the concentration of nisin using Micrococcus luteus as the indicator microorganism. A finite element computational model based on Fick's second law of diffusion was used to predict the radius of the inhibition zone in this diffusion bioassay. The model developed was used to calculate nisin concentration profiles as a function of time and position within the agar. The minimum inhibitory concentration (MIC) of nisin against M. luteus was determined experimentally. The critical time (T c) for growth of M. luteus within the agar diffusion bioassay was experimentally determined using incubation studies with nisin. The radius of the inhibition zone was predicted from the computational model as the location where the predicted nisin concentration at T c was equal to MIC. The MIC was experimentally determined to be 0.156 μg mL(-1), and T c was determined to be 7 h. Good agreement (R (2) = 0.984) was obtained between model-predicted and experimentally determined inhibition zone radii.

Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.

Listeria monocytogenes was recently found to enter a long-term-survival (LTS) phase, which may help explain its persistence in natural environments and within food processing plants. The purpose of this study was to investigate the effects of initial cell density, initial pH and type of broth (fresh vs. spent) on the transition of L. monocytogenes to the LTS phase and model the change in viable population density with time. Initial cell density (~10(6)-~10(10)CFU/ml) and initial pH (5.36-6.85) both significantly affected the transition of L. monocytogenes to the LTS phase (P<0.001) with initial cell density being the main determining factor. In contrast, type of broth did not significantly affect cell density change during the transition of stationary-phase cells at high initial density to the LTS phase (P>0.05). After 30-d incubation no significant differences in cell densities were observed between either type of broth or between any of the initial cell density/pH treatment combinations (P>0.05), where the mean viable cell density was 4.3±1.1×10(8)CFU/ml. L. monocytogenes responded to viable cell density in accordance with the logistic equation during transition to the LTS phase. The Agr quorum-sensing system does not appear to play a role in the transition to the LTS phase. Further research is needed to better understand the control mechanisms utilized by L. monocytogenes as it transitions to a coccoid, resistant and stable density state in the LTS phase., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Listeria monocytogenes is a foodborne pathogen of significant concern to the agricultural and food processing industry because of its ability to grow and persist in cool and moist environments and its association with listeriosis, a disease with a very high mortality rate. Although there have been no listeriosis outbreaks attributed to fresh mushrooms in the United States, retail surveys and recalls are evidence that L. monocytogenes contamination of mushrooms (Agaricus bisporus) can occur. The objective of this study was to determine the prevalence of Listeria spp., including L. monocytogenes, in a small-scale mushroom production facility on the campus of the Pennsylvania State University in the United States. Of 184 samples taken from five production zones within the facility, 29 (15.8%) samples were positive for Listeria spp. Among the Listeria spp. isolates, L. innocua was most prevalent (10.3%) followed by L. welshimeri (3.3%), L. monocytogenes (1.6%), and L. grayi (0.5%). L. monocytogenes was recovered only from the phase I raw material composting area. Isolates of L. monocytogenes were confirmed and serotyped by multiplex PCR. The epidemiological relatedness of the three L. monocytogenes isolates to those serotypes or lineages frequently encountered in listeriosis infections was determined by multi-virulence-locus sequence typing using six virulence genes, namely, prfA, inlB, inlC, dal, clpP, and lisR. The phylogenetic positions of the three isolates in the dendrogram prepared with data from other isolates of L. monocytogenes showed that all isolates were grouped with serotype 4a, lineage IIIA. To date, this serotype has rarely been reported in foodborne disease outbreaks.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major threat to human populations worldwide. Knowing the extent of MRSA genetic diversity within a healthcare facility may provide important insights into the epidemiology of this important pathogen. MRSA isolates recovered from nasal swabs of patients entering the Intensive Care Unit of the Penn State Milton S. Hershey Medical Center, USA, from 2008 to 2009 were genotyped using Staphylococcal Cassette Chromosome mec (SCCmec), multilocus sequence typing (MLST) and a newly developed multi-virulence-locus sequence typing (MVLST) scheme. Sequence data for seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqiL) and six virulence genes (alt, essC, geh, hlgA, htrA and sdrC) were used for MLST and MVLST analyses, respectively. MLST identified 12 sequence types (STs) within the hospital isolates. One ST designated ST5 was the most common subtype (38.8%) followed by ST105 (22.4%) and ST8 (16.4%). In contrast, MVLST identified 29 STs (Virulence Types, VTs) from the same set of isolates, with VT6 (32.8%) being the predominant subtype followed by VT9 (8.9%) and VT2 (8.9%). Subsequent analysis of 25 MRSA isolates associated with an outbreak at a Pennsylvania state prison revealed all isolates were VT2 and SCCmec type IVa. These results suggest that a combination of MVLST and SCCmec typing may clarify the epidemiology of MRSA. Additional research with a more diverse set of strains and correlation with conventional epidemiologic data are needed to validate this new subtyping strategy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fifty-three Listeria monocytogenes isolates obtained from Gorgonzola cheese and previously characterized with biochemical typing, serotyping and pulsed field gel electrophoresis (PFGE), were analyzed in this study. Seven virulence-associated genes were selected (actA, inlC, inlJ, plcA, prfA, hlyA and iap) and their presence was investigated using PCR. All virulence-associated genes were detected in 51 isolates. One isolate did not show amplification of the inlC gene and one other isolate, previously mis-identified as L. monocytogenes probably due to atypical phenotypes, resulted negative by PCR for all virulence genes and was identified as Listeria innocua by 16S rRNA gene sequencing analysis. A multiplex PCR assay was used to evaluate the presence of markers specific for epidemic clones (ECs) of L. monocytogenes. The marker specific for the recently identified epidemic clone V (ECV) was detected in 38 of 43 (88%) of serotype 1/2a isolates. These findings suggest that Gorgonzola cheese can represent a significant source of L. monocytogenes and potential health risk for listeriosis as almost all isolates (94%) could be potentially virulent and that 38 (~72%) were presumptive positive ECV. PCR screening for both virulence-associated genes and EC markers may be useful for rapidly evaluating the epidemic potential and public health risk posed by L. monocytogenes in PDO Gorgonzola cheese and other dairy products., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) each identified a diverse population of Listeria monocytogenes isolates, and within that, both methods had congruent subtypes that substantiated a predominant clone (clonal complex 8; virulence type 59; proposed epidemic clone 5 [ECV]) that has been causing human illness across Canada for more than 2 decades.

A novel multiplex PCR was developed which targeted virulence genes associated with the major clonal complexes (CCs) of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) in the USA. Most isolates (40/66) were identified as CC 5, while remaining isolates represented CCs 1, 8, 30, 45, 59, 133, and five isolates were not S. aureus., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Listeria monocytogenes can change its cellular morphology from bacilli to cocci during the transition to the long-term-survival (LTS) phase. The LTS cells demonstrated increased baro- and thermotolerance compared to their vegetative counterparts. So far, the underlying mechanisms that trigger this morphological and physiological transition remain largely unknown. In this study, we compared the transcriptomic profiles of L. monocytogenes serotype 4b strain F2365 at different growth stages in tryptic soy broth with yeast extract (TSBYE) using a whole-genome DNA chip approach. We identified a total of 225 differentially expressed genes (≥4-fold; P < 0.05) during the transition to the LTS phase in TSBYE. Genes related to cell envelope structure, energy metabolism, and transport were most significantly upregulated in the LTS phase. The upregulation of compatible solute transporters may lead to the accumulation of cellular solutes, lowering intracellular water activity and thus increasing bacterial stress resistance during the transition to the LTS phase. The downregulation of genes associated with protein synthesis may indicate a status of metabolic dormancy of the LTS cells. The transcriptomic profiles of resuscitated LTS cells in fresh TSBYE resembled those of log-phase cells (r=0.94), as the LTS cells rapidly resume metabolic activities and transit back to log phase with decreased baro- and thermotolerance.

A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.

by Philip E. Nelson, 2007 World Food Prize Laureate; Professor Emeritus, Food Science Dept., Purdue Univ. Just as society has evolved over time, our food system has also evolved over centuries into a global system of immense size and complexity. The commitment of food science and technology professionals to advancing the science of food, ensuring a safe and abundant food supply, and contributing to healthier people everywhere is integral to that evolution. Food scientists and technologists are versatile, interdisciplinary, and collaborative practitioners in a profession at the crossroads of scientific and technological developments. As the food system has drastically changed, from one centered around family food production on individual farms and home food preservation to the modern system of today, most people are not connected to their food nor are they familiar with agricultural production and food manufacturing designed for better food safety and quality. The Institute of Food Technologists-a nonprofit scientific society of individual members engaged in food science, food technology, and related professions in industry, academia, and government-has the mission to advance the science of food and the long-range vision to ensure a safe and abundant food supply contributing to healthier people everywhere. IFT convened a task force and called on contributing authors to develop this scientific review to inform the general public about the importance and benefits of food science and technology in IFT's efforts to feed a growing world. The main objective of this review is to serve as a foundational resource for public outreach and education and to address misperceptions and misinformation about processed foods. The intended audience includes those who desire to know more about the application of science and technology to meet society's food needs and those involved in public education and outreach. It is IFT's hope that the reader will gain a better understanding of the goals or purposes for various applications of science and technology in the food system, and an appreciation for the complexity of the modern food supply. Abstract:  This Institute of Food Technologists scientific review describes the scientific and technological achievements that made possible the modern production-to-consumption food system capable of feeding nearly 7 billion people, and it also discusses the promising potential of ongoing technological advancements to enhance the food supply even further and to increase the health and wellness of the growing global population. This review begins with a historical perspective that summarizes the parallel developments of agriculture and food technology, from the beginnings of modern society to the present. A section on food manufacturing explains why food is processed and details various food processing methods that ensure food safety and preserve the quality of products. A section about potential solutions to future challenges briefly discusses ways in which scientists, the food industry, and policy makers are striving to improve the food supply for a healthier population and feed the future. Applications of science and technology within the food system have allowed production of foods in adequate quantities to meet the needs of society, as it has evolved. Today, our production-to-consumption food system is complex, and our food is largely safe, tasty, nutritious, abundant, diverse, convenient, and less costly and more readily accessible than ever before. Scientific and technological advancements must be accelerated and applied in developed and developing nations alike, if we are to feed a growing world population., (© 2010 Institute of Food Technologists®.)

DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping Escherichia coli O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that rhs genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of E. coli O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7.

Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes were investigated. For part 1 of this analysis, L. monocytogenes ATCC 19115 was grown to log, stationary, death, and long-term-survival phases at 35 degrees C in tryptic soy broth with yeast extract (TSBYE). Cells were diluted in whole milk that had been subjected to ultrahigh temperatures (UHT whole milk) and then high-pressure processed (HPP) at 400 MPa for 180 s or thermally processed at 62.8 degrees C for 30 s. As cells transitioned from the log to the long-term-survival phase, the D(400 MPa) and D(62.8 degrees C) values increased 10- and 19-fold, respectively. Cells decreased in size as they transitioned from the log to the long-term-survival phase. Rod-shaped cells transitioned to cocci as they entered the late-death and long-term-survival phases. L. monocytogenes strains F5069 and Scott A showed similar results. For part 2 of the analysis, cells in long-term-survival phase were centrifuged, suspended in fresh TSBYE, and incubated at 35 degrees C. As cells transitioned from the long-term-survival phase to log and the stationary phase, they increased in size and log reductions increased following HPP or heat treatment. In part 3 of this analysis, cells in long-term-survival phase were centrifuged, suspended in UHT whole milk, and incubated at 4 degrees C. After HPP or heat treatment, similar results were observed as for part 2. We hypothesize that cells of L. monocytogenes enter a dormant, long-term-survival phase and become more barotolerant and thermotolerant due to cytoplasmic condensation when they transition from rods to cocci. Further research is needed to test this hypothesis and to determine the practical significance of these findings.

Previous molecular subtyping studies have defined four epidemic clones (ECs) of Listeria monocytogenes (ECI, ECII, ECIII, and ECIV). Partial sequences of eight virulence genes were previously shown to be identical within individual ECs of L. monocytogenes. The present study was conducted to determine if the sequences of other virulence genes and virulence gene regions are also conserved within these ECs. Six additional virulence genes--bsh, hly, inlJ, IplA1, pgdA, and srtA--and three additional virulence gene regions of actA, inlA, and inlB were selected based on their role in L. monocytogenes virulence, and intragenic regions of each gene were sequenced. Sequencing was performed on a diverse set of 44 to 48 L. monocytogenes strains. Results demonstrated that the sequenced regions of the nine virulence genes were identical within each of the ECs, and 257 new single nucleotide polymorphism (SNPs) were identified. ECIII (lineage II) was easily distinguishable from the other ECs, as 238 SNPs were observed in ECIII due to its significant evolutionary divergence from lineage I. With regard to the other ECs, there were 5 SNPs that represented an informative set, since these SNPs were able to differentiate specific ECs from all other unrelated strains used in this study. This study confirms our previous finding that virulence gene sequences are highly conserved within individual ECs and contain stable SNPs that can be used to very accurately differentiate ECs of L. monocytogenes from each other and from other diverse strains.

The aim of this study was to investigate the effect of water activity (aw) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various aws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 microg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of beta-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P<0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a approximately 7.5-log(10) reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P<0.05) when the peptone water concentration was decreased below 60% (aw approximately 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r2=0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low aw results in protein stabilization, which prevents protein denaturation and cell death during HPP.

A fragment end ligation-mediated PCR strategy was used to analyze the AscI pulsed-field gel electrophoresis patterns of Listeria monocytogenes epidemic clone II (ECII), which led to the identification of single-nucleotide polymorphisms (SNPs) in prophage regions that differentiated the two ECII outbreak clones. SNPs in prophages that differentiated the outbreak clones of ECIII and -IV were also identified.

The aim of this study was to investigate the effect of heat shock on the resistance of Listeria monocytogenes to high pressure processing (HPP). L. monocytogenes ATCC 19115 was grown to stationary phase at 15 degrees C and inoculated into whole ultrahigh-temperature milk at approximately 10(7) CFU/ml. Milk samples (5 ml) were placed into plastic transfer pipettes, which were heat sealed and then heated in a water bath at 48 degrees C for 10 min. Immediately after heat shock, the milk was cooled in water (20 degrees C) for 25 min and then placed on ice. The samples were high pressure processed at ambient temperature (approximately 23 degrees C) at 400 MPa for various times up to 150 s. Following HPP, the samples were spread plated on tryptic soy agar supplemented with yeast extract. Heat shock significantly increased the D400 MPa-value of L. monocytogenes from 35 s in non-heat-shocked cells to 127 s in heat-shocked cells (P < 0.05). Addition of chloramphenicol before heat shock eliminated the protective effect of heat shock (P < 0.05). Heat shock for 5, 10, 15, or 30 min at 48 degrees C resulted in maximal barotolerance (P < 0.05); increasing the time to 60 min significantly decreased survival compared with that at 5, 10, 15, or 30 min (P < 0.05). These results indicate that prior heat shock significantly increases the barotolerance of L. monocytogenes and that de novo protein synthesis during heat shock is required for this enhanced barotolerance.

Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.

A multiplex PCR assay which combines detection of bacteria of the genus Listeria, Listeria monocytogenes serotypes 1/2a and 4b, and epidemic clones I, II, and III of L. monocytogenes was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.

The aim of this study was to explore the effect of a wide range of growth temperatures, growth phases and plating media on the inactivation of Listeria monocytogenes by high pressure processing (HPP). In part one, L. monocytogenes was grown to mid-stationary phase at 4, 15, 25, 35 or 43 degrees C, inoculated into whole UHT milk at approximately 10(7) CFU/ml and high pressure processed at 400 MPa at room temperature (20-25 degrees C). Afterward, the HPP milk was plated on Tryptic Soy Yeast Extract Agar (TSYEA) and Modified Oxford Agar (MOX) to determine the degree of injury. For part two, cells were grown to mid-exponential, late-exponential or mid-stationary phase at 15 or 43 degrees C and processed in the same way. Time to reach a 5-log reduction was determined and data were analysed by ANOVA. The results from part one showed that both growth temperature and plating medium had a significant effect (P < 0.001) on the inactivation of stationary phase L. monocytogenes by HPP. Tukey's pairwise comparisons revealed that the effects of all temperatures, except 35 and 43 degrees C, were significantly different (P < 0.05). Cells grown at 15 degrees C were most sensitive to HPP, followed by cells grown at 4, 25 or 35 degrees C, with cells grown at 43 degrees C appearing to be the most resistant. Inactivation of cells grown at 4, 15 or 25 degrees C followed first order kinetics, whereas cells grown at 35 or 43 degrees C displayed non-linear inactivation kinetics due to tailing. In part two, both growth phase and plating medium had significant effects on the inactivation (P < or = 0.001) of L. monocytogenes by HPP. Cells grown at 15 degrees C to mid-stationary phase were the most pressure-resistant when tested on both media, and were significantly more resistant (P < 0.05) than cells grown at the same temperature to the other two phases of growth. There was no significant difference between mid- and late-exponential phase cells grown at 15 degrees C. When cells were grown at 43 degrees C, mid-exponential phase cells were significantly more sensitive (P < 0.05) than either late-exponential or mid-stationary phase cells, with no difference between late-exponential or mid-stationary phase cells. It was postulated that membrane composition, stationary phase proteins and/or stress proteins may affect pressure resistance.

A recently developed multi-virulence-locus sequence typing (MVLST) method showed improved discriminatory power for subtyping genetically diverse Listeria monocytogenes isolates and identified epidemic clone II isolates associated with two recent U.S. multistate listeriosis outbreaks. To evaluate the ability of MVLST to distinguish other epidemic clones and outbreak strains of L. monocytogenes, 58 outbreak-related isolates from 14 outbreaks and 49 unrelated isolates were analyzed. Results showed that MVLST provided very high discriminatory power (0.99), epidemiological concordance (1.0), stability, and typeability. MVLST accurately identified three previously known epidemic clones (epidemic clones I, II, and III) and redefined another epidemic clone (epidemic clone IV) in serotype 4b of L. monocytogenes. A set of 28 single nucleotide polymorphisms (SNPs) differentiated all epidemiologically unrelated isolates. A subset of 16 SNPs differentiated all epidemic clones and outbreak strains. Phylogenetic analysis showed congruence between MVLST clusters, serotypes, and previously defined genetic lineages of L. monocytogenes. SNPs in virulence genes appear to be excellent molecular markers for the epidemiological investigation of epidemics and outbreaks caused by L. monocytogenes.

A study was conducted to understand the descriptive and molecular epidemiology of antimicrobial-resistant gram-negative enteric bacteria in the feces of healthy lactating dairy cattle. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, spectinomycin, and tetracycline were isolated from the feces of 35, 8, 5, and 42% of 213 lactating cattle on 74, 39, 9, 26, and 82% of 23 farms surveyed, respectively. Antimicrobial-resistant gram-negative bacteria accounted for 5 (florfenicol) to 14% (tetracycline) of total gram-negative enteric microflora. Nine bacterial species were isolated, of which Escherichia coli (87%) was the most predominant species. MICs showing reduced susceptibility to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline were observed in E. coli isolates. Isolates exhibited resistance to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). Multidrug resistance (> or =3 to 6 antimicrobials) was seen in 40% of E. coli isolates from healthy lactating cattle. Of 113 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 93% of isolates, while the remaining 7% isolates carried the tet(A) determinant. DNA-DNA hybridization assays revealed that tet determinants were located on the chromosome. Pulsed-field gel electrophoresis revealed that tetracycline-resistant E. coli isolates (n = 99 isolates) belonged to 60 subtypes, which is suggestive of a highly diverse population of tetracycline-resistant organisms. On most occasions, E. coli subtypes, although shared between cows within the herd, were confined mostly to a dairy herd. The findings of this study suggest that commensal enteric E. coli from healthy lactating cattle can be an important reservoir for tetracycline and perhaps other antimicrobial resistance determinants.

This study was conducted to determine the penetration of 5% trisodium phosphate solution at various depths within punctures and calyces of apples spot inoculated with Escherichia coli O157:H7 and the effect of solution agitation on destruction of the pathogen. Sanitizer solutions containing radiolabeled disodium phosphate (DSP32) were able to penetrate apple tissues through punctures and calyx cavities. However, agitation of the solutions did not result in significantly greater penetration in these areas (P > 0.05). Overall, there were 1.57- and 1.1-log reductions of pathogen cells within 4-h-old punctures treated with and without trisodium phosphate solution agitation, respectively. Sanitizer solutions were effective in destroying pathogen cells residing within the upper 4.2-mm region of the punctures. Destruction of pathogen cells within open and closed calyces occurred mainly within the basin and the upper 3 mm of the calyx cavity. Treatment with agitated sanitizer solution resulted in a 0.67-log reduction in pathogen concentration within open calyces. In contrast, treatment of closed calyces resulted in a 1.37-log reduction, mainly within the basin. Washing with water alone appeared to result in further penetration of the cells within calyces without significantly reducing the number of pathogen cells (P > 0.05). To develop more effective methods for reducing contamination on produce, it is important to know the extent of sanitizer penetration and its effect on destruction of pathogens.

The ability of Escherichia coli O157:H7 to penetrate and grow within punctures, fresh-cut surfaces, and calyces of Golden Delicious apples was investigated. A three-strain cocktail of E. coli O157:H7 resistant to ampicillin was used to inoculate fresh and 48-h-old punctures, fresh-cut surfaces, and open or closed calyces. A concentric cutting procedure was used to evaluate depth of penetration within punctures and prevent cross contamination during sampling. Within 2 h, E. coli O157:H7 penetrated vertically through the fresh punctures and 3.4 mm within the underlying parenchyma. After 48 h, E. coli O157: H7 cells penetrated up to 5.5 mm within the punctures and >2.6 mm horizontally away from fresh punctures. However, 48-h-old punctures did not permit penetration beyond their boundaries. Fresh-cut surfaces permitted up to 2.8 mm penetration after 24 h. Onset of growth of E. coli O157:H7 occurred 4 to 8 h postinoculation on fresh punctures and fresh-cut surfaces with populations increasing by 3 logs after 48 h. E. coli O157:H7 penetrated within calyces regardless of the extent of opening or method of inoculation. However, E. coli O157:H7 was never recovered from the inner core of apples. Computed tomography scan imaging revealed that closed calyces effectively prevented penetration of sodium iodide solutions within the calyx cavity. Lack of solution penetration may explain why sanitizing treatments are ineffective in inactivating microbial cells within the calyx. Understanding the role of morphological differences in permitting or restricting bacterial penetration may lead to development of more effective strategies to enhance the safety of fresh horticultural products.

Multi-virulence-locus sequence typing (MVLST) was used to analyze isolates from two major listeriosis outbreaks in the United States in 1998 and 2002 that were due to consumption of contaminated hot dogs and turkey deli meat, respectively. MVLST demonstrated high epidemiological relevance and indicated that the two outbreaks were the result of one epidemic.

Listeria monocytogenes serotypes 1/2a and 4b are responsible for the majority of cases of human listeriosis worldwide. In this study, a multiplex PCR assay was developed to allow rapid identification and easily interpretable differentiation of serotypes 1/2a and 4b from other serotypes of L. monocytogenes by simultaneously targeting two virulence genes (inlB and inlC) and two serotype-specific genes (ORF2372 and Imo0171). A subsequent gel extraction and sequence typing analysis of the highly polymorphic intragenic regions in inlB and inlC simplified a previously developed multi-virulence-locus sequence typing scheme and provided discriminatory power for subtyping L. monocytogenes similar to pulsed-field gel electrophoresis analysis.

A five-isolate cocktail of Listeria monocytogenes (10(3) cfu/ml in skim or whole raw milk) was subjected to 450 MPa for 900 s or 600 MPa for 90 s. The effects of prior growth temperature, type of milk (skim vs. whole), type of recovery-enrichment media (optimized Penn State University [oPSU] broth, Listeria Enrichment Broth [LEB], Buffered LEB [BLEB], Modified BLEB [MBLEB], and milk), storage temperature and storage time on the recovery of L. monocytogenes were examined. Optimized PSU broth significantly increased the recovery of L. monocytogenes following high pressure processing (HPP), and was 63 times more likely to recover L. monocytogenes following HPP, compared to LEB, BLEB and MBLEB broths (p<0.05; Odds Ratio=63.09, C.I. 23.70-167.96). There was a significant main effect for prior growth temperature (p<0.05). However, this relationship could not be interpreted given the significant interaction effects between temperature and both pressure and milk type. HPP-injured L. monocytogenes could be recovered using both LEB and oPSU broths after storage of milk at 4, 15 and 30 degrees C, with recovery being maximal after 24 to 72 h of storage; however, recovery yield dropped to 0% after prolonged storage of milk at 4 and 30 degrees C. In contrast, storage of milk at 15 degrees C yielded the most rapid rate of recovery and the highest recovery yield (100%), which remained high throughout the 14 days of storage at 15 degrees C. The above factors need to be taken into consideration when designing challenge studies to insure complete inactivation of L. monocytogenes and possibly other foodborne pathogens during high pressure processing of foods.

During commercial processing, eggs are washed in an alkaline detergent and then rinsed with chlorine to reduce dirt, debris, and microorganism levels. The alkaline and acidic fractions of electrolyzed oxidizing (EO) water have the ability to fit into the 2-step commercial egg washing process easily if proven to be effective. Therefore, the efficacy of EO water to decontaminate Salmonella Enteritidis and Escherichia coli K12 on artificially inoculated shell eggs was investigated. For the in vitro study, eggs were soaked in alkaline EO water followed by soaking in acidic EO water at various temperatures and times. Treated eggs showed a reduction in population between > or = 0.6 to > or =2.6 log10 cfu/g of shell for S. Enteritidis and > or =0.9 and > or =2.6 log10 for E. coli K12. Log10 reductions of 1.7 and 2.0 for S. Enteritidis and E. coli K12, respectively, were observed for typical commercial detergent-sanitizer treatments, whereas log10 reductions of > or =2.1 and > or =2.3 for S. Enteritidis and E. coli K12, respectively, were achieved using the EO water treatment. For the pilot-scale study, both fractions of EO water were compared with the detergent-sanitizer treatment using E. coli K12. Log10 reductions of > or = 2.98 and > or = 2.91 were found using the EO water treatment and the detergent-sanitizer treatment, respectively. The effects of 2 treatments on egg quality were investigated. EO water and the detergent-sanitizer treatments did not significantly affect albumen height or eggshell strength; however, there were significant affects on cuticle presence. These results indicate that EO water has the potential to be used as a sanitizing agent for the egg washing process.

The BAX PCR for screening Listeria monocytogenes is a commercial PCR assay for specifically targeting L. monocytogenes, a foodborne pathogen that can contaminate a variety of foods and cause a potentially fatal disease, listeriosis, among high-risk populations. The high specificity (> 98%) of this PCR assay is achieved by targeting a species-specific genomic region (approximately 400 bp) presumably found only in L. monocytogenes. In this study, the identity of the BAX PCR-targeted genomic region was determined by using PCR cloning, DNA sequencing, and basic local alignment search tool (BLAST) analysis of the amplicon sequences of an L. monocytogenes serotype 1/2a strain. BLAST analysis identified the BAX PCR amplicon (GenBank accession no. AY364605) as a 423-bp genomic region between nucleotides 224,409 and 224,831 in the genome of L. monocytogenes (serotype 1/2a strain EGD-e), including a 145-bp noncoding region and a 278-bp partial coding sequence of a putative gene, lmo2234. The translated amino acid sequence (92 amino acids) of this partial coding region is highly conserved between L. monocytogenes and Listeria innocua (93% homology). Reverse-position-specific BLAST analysis identified a conserved domain in Lmo2234 that was similar (95.3% aligned, E value = 9E-18) to the consensus amino acid sequence of sugar phosphate isomerases/epimerases (National Center for Biotechnology Information conserved domain database accession no. COG 1082.1, IolE), indicating that Lmo2234 might be involved in bacterial carbohydrate transport and metabolism.

A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes.

A one-step, recovery-enrichment broth, optimized Penn State University (oPSU) broth, was developed to consistently detect low levels of injured and uninjured Listeria monocytogenes cells in ready-to-eat foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrated to the surface, forming a black zone. This migration separated viable from nonviable cells and the food matrix, thereby reducing inhibitors that prevent detection by molecular methods. The high Listeria-to-background ratio in the black zone resulted in consistent detection of low levels of L. monocytogenes in pasteurized foods by both cultural and molecular methods, and greatly reduced both false-negative and false-positive results. oPSU broth does not require transfer to a secondary enrichment broth, making it less laborious and less subject to external contamination than 2-step enrichment protocols. Addition of 150mM D-serine prevented germination of Bacillus spores, but not the growth of vegetative cells. Replacement of D-serine with 12 mg/L acriflavin inhibited growth of vegetative cells of Bacillus spp. without inhibiting recovery of injured Listeria cells. oPSU broth may allow consistent detection of low levels of injured and uninjured cells of L. monocytogenes in pasteurized foods containing various background microflora.

Mixed raw egg contents were inoculated with approximately 10 CFU of Salmonella Enteritidis and supplemented with 0 to 7 mg of FeSO4 per g of egg contents. Egg contents were then incubated at 37 degrees C, and Salmonella Enteritidis colonies were enumerated for up to 106 h. Iron supplementation significantly enhanced the growth of Salmonella Enteritidis. Within the first 24 h of incubation, the optimum iron level for Salmonella Enteritidis growth in egg contents was between 0.2 and 2 mg of FeSO4 per g of egg contents. After 24 h of incubation at 37 degrees C. Salmonella Enteritidis counts in eggs supplemented with 0.5 mg of FeSO4 per g of egg contents consistently reached approximately 1 x 10(9) CFU/ml, whereas Salmonella Enteritidis counts in eggs without iron supplementation varied from less than 5 CFU/ml to 8.4 x 10(6) CFU/ml. A 3 by 3 factorial design was used to study the effect of type of preenrichment and level of iron supplementation on the growth of Salmonella Enteritidis in egg contents. No significant differences in Salmonella Enteritidis counts between preenrichment and nonpreenrichment treatments were observed when egg contents were supplemented with 0.5 mg of FeSO4 per g of egg contents. It was concluded that preenrichment was not necessary for isolation of Salmonella Enteritidis from eggs. The effect of iron supplementation on the sensitivity of detection by the direct plating method was investigated. The direct plating method detected a significantly higher percentage of Salmonella Enteritidis in raw egg contents supplemented with 0.5 mg of FeSO4 per g of egg contents (90%) than in raw egg contents without iron supplementation (63.3%).

Due to undesirable quality changes, Lebanon bologna is often processed at temperatures that do not exceed 48.8 degrees C (120 degrees F). Therefore, it is important to study parameters that influence the destruction of Escherichia coli O157:H7 in Lebanon bologna. The objective of the present study was to determine the influence of curing salts (NaCl and NaNO2) on the destruction of E. coli O157:H7 during Lebanon bologna processing. Fermentation to pH 4.7 at 37.7 degrees C reduced populations of E. coli O157:H7 by approximately 0.3 log10, either in the presence or absence of curing salts. Subsequent destruction of E. coli O157:H7 during heating of fermented product to 46.1 degrees C was significantly reduced by the presence of 3.5% NaCl and 156 ppm NaNO2, compared to product without curing salts (P < 0.01). The presence of a higher level of NaCl (5%) in Lebanon bologna inhibited the growth of lactic acid bacteria (LAB), which yielded product with higher pH (approximately 5.0) and significantly reduced the destruction of E. coli O157:H7 even further (P < 0.05). Lower concentrations of NaCl (0, 2.5%) yielded Lebanon bologna with higher LAB counts and lower pHs, compared to product with 5% NaCl. When lactic acid was used to adjust pH in product containing different levels of NaCl, it was determined that low pH was directly influencing destruction of E. coli O157:H7, not NaCl concentration.

Escherichia coli O157:H7, Salmonella Typhimurium, or Listeria monocytogenes was spread onto the surface of Lebanon bologna luncheon slices using sterile glass rods. The inoculated slices were stacked and vacuum packaged. The packages were stored at 3.6 or 13 degrees C. The foodborne pathogens. E. coli O157:H7, Salmonella Typhimurium, or L. monocytogenes were reduced in Lebanon bologna during storage at 3.6 or 13 degrees C. The higher storage temperature (13.0 degrees C) resulted in significantly faster destruction of E. coli O157:H7 and L. monocytogenes, compared to storage at refrigeration temperature (3.6 degrees C) (P < 0.005). E. coli O157:H7 was the most resistant to destruction among the three foodborne pathogens. A linear destruction of E. coli O157:H7 occurred only after an initial lag period. Storage temperature did not have a significant effect on the rate of destruction of Salmonella Typhimurium. Foodborne pathogens inoculated prior to fermentation did not show any enhanced survival compared to control cells (inoculated after fermentation) during storage of the Lebanon bologna at 3.6 degrees C.

Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip. Different combinations of incubation temperatures and lithium, magnesium, and D-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L. monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora. A concentration of 212 mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L. monocytogenes. A concentration of 15.8 mM MgSO4 was found to be optimum for the recovery and detection of L. monocytogenes. A concentration of 140.2 mM D-serine was found to completely inhibit the germination of Bacillus subtilis var. globii spores but not recovery and detection of L. monocytogenes. Under optimum concentrations of LiCl, MgSO4, and D-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28 degrees C was determined to be optimal. In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30 degrees C was found to be optimal. Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio. This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L. monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).

Recently, numerous product recalls and one devastating outbreak that claimed 21 lives were attributed to Listeria monocytogenes in ready-to-eat meat products. Consequently, the Food Safety and Inspection Service published a federal register notice requiring manufacturers of ready-to-eat meat and poultry products to reassess their hazard analysis and critical control point plans for these products as specified in 9 CFR 417.4(a). Lebanon bologna is a moist, fermented ready-to-eat sausage. Because of undesirable quality changes. Lebanon bologna is often not processed above 48.9 degrees C (120 degrees F). Therefore, the present research was conducted to validate the destruction of L. monocytogenes in Lebanon bologna batter in a model system. During production, fermentation of Lebanon bologna to pH 4.7 alone significantly reduced L. monocytogenes by 2.3 log10 CFU/g of the sausage mix (P < 0.01). Heating the fermented mix to 48.9 degrees C in 10.5 h destroyed at least 7.0 log10 CFU of L. monocytogenes per g of sausage mix. A combination of low pH (5.0 or lower) and high heating temperatures (> or =43.3 degrees C, 115 degrees F) destroyed more than 5 log10 CFU of L. monocytogenes per g of sausage mix during the processing of Lebanon bologna. In conclusion, an existing commercial process, which was validated for destruction of Escherichia coli O157:H7, was also effective for the destruction of more than 5 log10 CFU of L. monocytogenes.

Various primary enrichment broths, including University of Vermont medium (UVM), Listeria enrichment broth (LEB), modified LEB, and aerobic and anaerobic L-PALCAMY, were compared with aerobic and anaerobic Pennsylvania State University (PSU) broths for the detection of severely heat-injured (62.8 degrees C for 5, 10, or 15 min; no colony appearance after heat injury on aerobic Trypticase soy agar containing 0.6% yeast extract and modified Oxford medium) Listeria monocytogenes Scott A. Anaerobic conditions were produced by adding L-cysteine and then purging the headspace with N2. The effect of uninjured background flora (10(3) CFU/ml of Enterococcus faecium) on frequency of detection was examined. Anaerobic PSU broth resulted in the lowest false-positive rate and the highest frequency of detection of severely heat-injured L. monocytogenes compared with UVM, LEB, and modified LEB (P < 0.05). The presence of E. faecium significantly enhanced the detection of heat-injured (10 min at 62.8 degrees C) L. monocytogenes in aerobic and anaerobic PSU and aerobic and anaerobic L-PALCAMY broths (P < 0.05). The highest concentration of uninjured E. faecium (>10(6) CFU/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). A heat-resistant, LiCl-tolerant Lactobacillus isolate from raw milk increased the rate of both false-positive and false-negative reactions.

The effect of different reducing agents (L-cysteine, Oxyrase, and Enterococcus faecium) and their combinations on the detection of heat-injured (62.8 degrees C, 7.5 min or 10 min) Listeria monocytogenes was examined. The incorporation of L-Cysteine (0.5 g/liter) yielded higher percentage detection than any of the other reducing agents (P < 0.05). The combination of Oxyrase (10 U/ml) and E. faecium (10(3) CFU/ml) synergistically enhanced the detection of L. monocytogenes heat-injured for 10 min at 62.8 degrees C (P < 0.05). Simultaneous addition of L-cysteine (0.5 g/liter) and Oxyrase (10 U/ml) significantly lowered the recovery of heat-injured L. monocytogenes (P < 0.05). Higher activities of Oxyrase (50 U/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). The rates of depletion of relative percentage O2 were in the order: L-cysteine (0.5 g/liter; 6.63%/ min) > Oxyrase (10 U/ml; 5.00%/min) > E. faecium (10(8) CFU/ml; 1.66%/min) > E. faecium (10(3) CFU/ml; 0.20%/min). The final levels of redox potential (Eh) achieved were -110.5 mV, -100 mV, -83.5 mV, and -25 mV for E. faecium (10(8) CFU/ml), L-cysteine, Oxyrase, and E. faecium (10(3) CFU/ml), respectively.

A simple anaerobic recovery-enrichment system, semisolid Penn State University (ssPSU) broth, that enhances recovery of heat-injured Listeria monocytogenes, was rapidly achieved in 10-ml screw-capped tubes by adding Bacto-agar (2.5 g/liter) and L-cysteine (0.5 g/liter) to Penn State University broth. Glucose was removed from the formulation for ssPSU broth to prevent the growth of thermoduric lactobacilli. Ferric ammonium citrate was added to ssPSU broth to detect esculin hydrolysis and to indicate the presumptive presence of L. monocytogenes. Replacement of phosphate buffer with 3-[N-morpholino]propanesulfonic acid (MOPS) buffer and addition of magnesium sulfate (15 mM) enhanced recovery and detection of L. monocytogenes heat treated at 62.8 degrees C for 20 min. D-Serine, at a concentration of 150 mM, was found to inhibit germination of Bacillus spp. spores but did not inhibit severely heat-injured L. monocytogenes. Finally, ssPSU broth was modified (to mPSU broth) to contain the following: (i) Bacto-agar, 2.5 g/liter; (ii) ferric ammonium citrate, 0.5 g/liter; (iii) MOPS buffer, pH 7.0; (iv) D-serine, 13.7 g/liter; (v) D-alanine, 11.6 g/liter; and (iv) magnesium sulfate, 1.81 g/liter. Incubation temperature significantly affected the recovery and detection of severely heat-injured L. monocytogenes. L. monocytogenes that were heat challenged in filter-sterilized whole milk at 62.8 degrees C for 20, 25, and 30 min could not be detected at incubation temperatures > or = 30 degrees C but were consistently detected after incubation at 25 degrees C for 174, 199, and 330 h, respectively. Heat-injured cells of L. monocytogenes that were added to various commercial brands of pasteurized whole milk were also detected using mPSU broth. When clostridial spores (10(4) spores per ml) were added to filter-sterilized milk containing either heat-injured or non-heat-injured L. monocytogenes, only the latter could be detected in mPSU broth. The mPSU broth system requires no purging with nitrogen gas to create anaerobic conditions and permits recovery, growth, and detection of L. monocytogenes in one vessel in the presence of thermoduric background microflora commonly found in pasteurized milk.

This study was undertaken to determine if association with collagen enables Escherichia coli O157:H7 to resist high-pH treatments and to determine the effects of high pH on the survival of E. coli O157:H7 within different layers of beef tissue. E. coli O157:H7 was inoculated onto purified bovine type I collagen on 12-mm2 circular glass coverslips, plain 12-mm2 circular glass coverslips (control), and 12-mm2 irradiated (cobalt-60) lean beef tissue. The rates of destruction of E. coli O157:H7 inoculated on coverslips in pH 10.5 NaHCO3-NaOH buffer at 35 degrees C were determined at various sampling times. E. coli O157:H7 cells associated with collagen and treated in the same manner were also examined using scanning electron microscopy to determine if association with collagen enabled the organism to resist high-pH treatments. The inoculated tissue was treated in pH 13.0 NaHCO3-NaOH buffer at 25 degrees C, and penetrating cells of E. coli O157:H7 were recovered using a cryostat technique. There was no significant difference (P < 0.05) between the rates of destruction of collagen-associated E. coli O157:H7 and non-collagen-associated E. coli O157:H7 following exposure to high-pH treatments. Scanning electron micrographs showed that collagen-associated E. coli O157:H7 cells appeared physically damaged by exposure to high-pH treatments, and association of E. coli O157:H7 to collagen did not increase the resistance of the organism to destruction by high-pH rinses. No significant differences were seen between 20 ml of NaHCO3-NaOH buffer at pH 13.0 (treatment) and 20 ml of distilled water at pH 7.0 (control) when E. coli O157:H7 cells were recovered in beef tissue at depths of up to 2,000 microm (P < 0.05). The ability of E. coli O157:H7 to penetrate beef tissue may be an important factor in reducing the effectiveness of high-pH treatments in killing this organism on beef tissue. This finding should be considered in the future when designing treatments to decontaminate beef carcasses.

Listeria monocytogenes, a psychrotrophic microorganism, has been the cause of several food-borne illness outbreaks, including those traced back to pasteurized fluid milk and milk products. This microorganism is especially important because it can grow at storage temperatures recommended for milk (< or =7 degrees C). Growth of L. monocytogenes in fluid milk depends to a large extent on the varying temperatures it is exposed to in the postpasteurization phase, i.e., during in-plant storage, transportation, and storage at retail stores. Growth data for L. monocytogenes in sterilized whole milk were collected at 4, 6, 8, 10, 15, 20, 25, 30, and 35 degrees C. Specific growth rate and maximum population density were calculated at each temperature using these data. The data for growth rates versus temperature were fitted to the Zwietering square root model. This equation was used to develop a dynamic growth model (i.e., the Baranyi dynamic growth model or BDGM) for L. monocytogenes based on a system of equations which had an intrinsic parameter for simulating the lag phase. Results from validation of the BDGM for a rapidly fluctuating temperature profile showed that although the exponential growth phase of the culture under dynamic temperature conditions was modeled accurately, the lag phase duration was overestimated. For an alpha0 (initial physiological state parameter) value of 0.137, which corresponded to the mean temperature of 15 degrees C, the population densities were underpredicted, although the experimental data fell within the narrow band calculated for extreme values of alpha0. The maximum relative error between the experimental data and the curve based on an average alpha0 value was 10.42%, and the root mean square error was 0.28 log CFU/ml.

Fermented meats have caused food-borne illness due to enterohemorrhagic Escherichia coli. Consumption of Lebanon bologna was epidemiologically associated wit a recent outbreak of salmonellosis. The present study was conducted to determine the effect of pH (after the fermentation step), final heating temperature, and time on destruction of E. coli O157:H7 and Salmonella typhimurium in Lebanon bologna. Raw Lebanon bologna mix was inoculate with either of the pathogens (ca.10(8) CFR/g and fermented for 12 h at 80 degrees F (26.7 degrees C) and then at 100 degrees F (37.8 degrees C) unit the pH reached wither 5.2 or 4.7. The mix was then heated to 110, 115, or 120 degrees F (43.3, 46.1, or 48.9 degrees C). The bologna was sampled at various times, decimally diluted, and plated on either McConkey sorbitol agar or XLD agar to enumerate E. coli O157:H7 and S. typhimurium, respectively. Fermentation alone reduced populations of both pathogens by < 2 log units and heating alone reduced populations of E. coli O157:H7 by < 3 log units. A combination of fermenting to either pH 5.2 or 4.7, followed by heating at 110 degrees F (43.3 degrees C) for 20h, 115 degrees F (46.1 degrees C) for 10 h, or 120 degrees F (48.9 degrees C) for 3 h reduced populations of both pathogens by > 7 log units. Overall S. typhimurium cells were either equally or significantly less resistant (P < 0.01) than cells of E. coli O157:H7. Significantly interactions (P < 0.01) among the three factors for the destruction of E. coli O157:H7 were observed. A process-specific regression equation was developed to predict the destruction of E. coli O157:H7 in Lebanon bologna.