La listeriosi és una malaltia infecciosa causada pel bacteri Listeria monocytogenes. Aquesta malaltia es transmet principalment per via digestiva a través dels aliments contaminats. L. monocytogenes es diferencia d’altres patògens per la seva alta capacitat de resistència a les condicions adverses i per presentar gran ubiqüitat en el medi ambient. Aquesta sèrie de factors afavoreix la contaminació dels aliments. La listeriosi segueix una tendència ascendent i està considerada com a una malaltia emergent. No obstant això, a Espanya, la listeriosi no està inclosa en el sistema de malalties de declaració obligatòria (MDO), fet que comporta la manca de vigilància epidemiològica. L’objectiu d’aquest treball és recollir de forma exhaustiva tota la informació epidemiològica disponible sobre la listeriosi a Espanya durant el període 2001-2007, per tal de conèixer la càrrega i l’evolució de la malaltia al nostre país i com a primer pas cap a la seva recollida sistemàtica i continuada. Per a desenvolupar aquest objectiu es va dissenyar un estudi descriptiu retrospectiu basat en la recollida de la informació per comunitats autònomes, segons una estratègia amb quatre etapes diferenciades: obtenció dels casos de listeriosis oficialment declarats; revisió de les pàgines webs oficials; contacte directe amb els responsables de vigilància epidemiològica a la recerca d’informació no publicada sobre listeriosi a la seva comunitat; i revisió de la literatura científica. Es va disposar de la informació de forma efectiva en 12 comunitats autònomes. A l’esmentat període es van recogir 1.242 casos de listeriosis, la qual cosa representa una taxa d’incidència de 0,56 casos per 100.000 habitants i any, que n’és molt superior a la oficialment declarada (0,16). Únicament el 1,78% dels casos estan associats a brots. En el periode esmentat la incidència té una tendència ascendent (p<0,001). La informació epìdemiològica recollida és molt escassa. La listeriosi és més freqüent en homes (59,75%) i a les etapes extremes de la vida (0,99 i 1,36 casos per 100.000 habitants/any en les persones >60 anys i 0-4 anys, respectivament). Un de cada cinc casos de listeriosis eren neonats o dones embarassades, que la meningitis i/o sepsis va ser la manifestació clínica més rellevant (superior al 50% dels casos) i que un 18% dels casos no tenia cap malaltia de base. La notificació dels casos va ser major als mesos centrals de l’any. Els aïllaments de L. monocytogenes van ser majoritaris en sang i/o líquid cefaloraquidi (93%), I el serotip més freqüent va ser el 4b (66,6%). La letalitat va ser del 21,3% en els 277 casos de listeriosis amb informació sobre el resultat clínic. La inclusió d’aquesta malaltia al sistema MDO permitria dimensionar la seva presència a Espanya, així com conèixer les característiques de l’afectació humana, ens igualaria a la resta de països de la Unió Europea, i permetria impulsar una prevenció i control eficaç. / La listeriosis es una enfermedad infecciosa causada por la bacteria Listeria monocytogenes. Esta enfermedad se transmite principalmente por vía digestiva, a través de los alimentos contaminados L. monocytogenes se diferencia de otros patógenos por su alta capacidad de resistencia a condiciones adversas y por presentar gran ubicuidad en el medio ambiente. Todos estos factores favorecen la contaminación de los alimentos. La listeriosis sigue una tendencia ascendente y está considerada como una enfermedad emergente. Sin embargo, en España, la listeriosis no está incluida en el sistema de Enfermedades de Declaración Obligatoria (EDO) lo que conlleva una falta de vigilancia epidemiológica. El objetivo del presente trabajo ha sido recoger de forma exhaustiva la información epidemiológica disponible sobre la listeriosis en España durante el periodo 2001-2007, para conocer la carga y evolución de la enfermedad en el país y como un primer paso hacia su recogida sistemática y continua. Para desarrollar este objetivo se diseñó un estudio descriptivo retrospectivo basado en la recogida de información por comunidades autónomas, según una estrategia con cuatro etapas diferenciadas: obtención de los casos de listeriosis oficialmente declarados; revisión de las páginas webs oficiales; contacto directo con los responsables de vigilancia epidemiológica en busca de información no publicada sobre listeriosis en su comunidad; y revisión de la literatura científica. Se obtuvo la información de forma efectiva en 12 Comunidades Autónomas. Para el periodo citado se recogieron 1.242 casos de listeriosis, lo que supone una tasa de incidencia media de 0,56 casos de 100.000 habitantes y año, siendo muy superior a la oficialmente declarada (0,16). Únicamente el 1,78% de los casos se asociaron a brotes, desconociéndose el origen de los alimentos implicados en todos los brotes. La incidencia en el periodo de estudio ha sido ascendente (p<0,001). La información epidemiológica obtenida ha sido escasa. La listeriosis es más frecuente en hombres (59,75%) y en las etapas distales de la vida (0,99 y 1,36 casos por 100.000 habitantes en personas >60 años y 0-4 años, respectivamente. Uno de cada cinco casos de listeriosis eran neonatos o mujeres gestantes, que la meningitis y/o sepsis fue la manifestación clínica más relevante (superior al 50% de los casos) y que un 18% de los casos no tenían ninguna enfermedad de base. La notificación de los casos fue mayor en los meses centrales del año. Los aislamientos de L. monocytogenes se realizaron mayoritariamente en sangre y/o líquido cefalorraquídeo (93%), y el serotipo más frecuente fue el 4b (66,6%). La letalidad fue del 21,3% en los 277 casos de listeriosis con información sobre el resultado clínico. La inclusión de esta enfermedad en el sistema EDO permitiría dimensionar su presencia en España, así como conocer las características de la afectación humana, nos igualaría al resto de países de la Unión Europea, e impulsar una prevención y control eficaz. / The listeriosis is an infectious disease caused by the bacterium Listeria monocytogenes. This disease is transmitted mainly by digestive tract through contaminated foods. L. monocytogenes difference of other pathogens by its high endurance to adverse conditions and having a great ubiquity in the environment. All these factors contribute to the food pollution. The listeriosis follows an ascending trend and is considered to be an emergent disease. However, in Spain the listeriosis is not included in the mandatory notification which leads to a lack of epidemiological surveillance. The aim of this work has been collect to comprehensively epidemiological information available on listeriosis in Spain during 2001-2007, to know the load and disease evolution in the country and as a first step towards systematic and continuous collection. To develop this aim it was designed a descriptive retrospective study based on the collection of information by autonomous communities, according to a strategy with four differentiated stages: obtaining of the cases of listeriosis officially declared; review of the official websites; direct contact with those epidemiological surveillance responsible for unpublished information on listeriosis in their community; and review of the scientific literature. We obtained the information effectively in 12 autonomous communities. For the above period were collected 1.242 cases of listeriosis, which is an average incidence rate of 0.56 cases for 100.000 inhabitants and year, still much higher than the officially declared (0.16). Only 1.78% of the cases were associated with outbreaks. During 2001-2007 the incidence has presented an upward trend (p<0.001). The epidemiological information obtained was limited. The listeriosis is more common in men (59.75%) and the extreme stages of life: 55.48% in persons >60 years and 16.20% in children between 0-4 years. The incidence rate was 0.99 and 1.36 cases for 100.000 inhabitants/year in persons >60 years and 0-4 years, respectively. One of each five cases of listeriosis were neonates or pregnant women, that the meningitis was the most important clinical manifestation (over 50% of cases) and that 18% of cases had not underlying disease. The notification of the cases was higher in the central months of the year. The isolates of L.monocytogenes were mainly realised in blood and/or cerebrospinal fluid (93%) and serotype 4b was the most frequent. The mortality rate was 21.3% in 227 cases of listeriosis with information about clinical result. The inclusion of this disease in the mandatory notification would allow to measure its presence in Spain, as well as to know the characteristics of the human infection, we would match the rest of European Union countries, and to promote effective prevention and control.

Listeria monocytogenes is a foodborne pathogenic bacterium responsible for ~500 deaths and a financial burden of ~$2.3 billion each year in the United States. Though a zero tolerance policy is enforced with regard to its detection in cooked ready-to-eat foods, additional preemptive control alternatives are required for certain products. Among these alternatives are strategies permitting the usage of food antimicrobial combinations to control the pathogen. Research on antimicrobial combinations can provide insight into more efficient control of the pathogen, but is currently lacking. The purpose of this study was to evaluate the in vitro inhibition of L. monocytogenes exposed to the antimicrobials e-Poly-L-Lysine (EPL), lauric arginate ester (LAE), and sodium lactate (SL) at pH 7.3, octanoic acid (OCT) at pH 5.0, and nisin (NIS) and acidic calcium sulfate (ACS) at both pH 5.0 and 7.3. A broth dilution assay was used to determine single antimicrobial minimum inhibitory and bactericidal concentrations for L. monocytogenes Scott A, 310, NADC 2783, and NADC 2045. Optical density differences (delta<0.05 at 630 nm) were used to denote inhibition. Concentrations producing population decreases of greater than or equal to 3.0 log10 CFU/ml after incubation were considered bactericidal. Inhibition resulting from combinations of antimicrobials (NIS+ACS, EPL+ACS, SL+ACS, NIS+LAE, OCT+ACS, and OCT+NIS) was assessed using a checkerboard assay, and fractional inhibitory concentrations (FIC) were determined. FIC values were plotted on isobolograms and were used to create FIC indices (FICI). Isobologram curvature was used to classify combinations as synergistic, additive, or antagonistic. From FIC indices, interactions were defined as antagonistic (FICI >1.0), additive (FICI =1.0), or synergistic (FICI &lt;1.0). Strain-dependent factors had a bearing on MIC and MBC values for NIS and EPL. At pH 7.3, NIS+ACS displayed synergistic inhibition, NIS+LAE and EPL+ACS demonstrated additive-type interactions, and the SL+ACS pairing was unable to be defined. At pH 5.0, interpretation of the OCT+NIS interaction also presented challenges, while the OCT+ACS combination resulted in synergistic behavior. Additional studies are needed to validate in vitro findings on surfaces of ready-to-eat meats. Future in vivo studies should investigate the ability of synergistic combinations (NIS+ACS and OCT+ACS) to control the pathogen. Better characterizations of inhibitory mechanisms should also be performed.

A partir de los años ochenta, el aumento de casos de listeriosis humana y su posible relación con alimentos contaminados, ha venido preocupando a las autoridades sanitarias de todo el mundo. Aunque en el Perú no hay reportes de una asociación entre listeriosis y alimentos contaminados, se ha informado sobre estudios que han identificado esta bacteria en productos hidrobiológicos frescos y procesados, leche cruda y sus derivados. El Objetivo del presente trabajo fue determinar la incidencia de Listeria monocytogenes en pollos y verduras obtenidos de diversos mercados y centros de abastecimiento de Lima. En total, se analizaron 100 muestras, de las cuales 50 fueron muestras de carne de pollo fresco y las otras 50 muestras fueron diversas verduras frescas (espárrago, col, apio, espinaca y lechuga). El análisis microbiológico se realizó de acuerdo a la metodología recomendada en el Bacteriological Analytical Manual de la FDA y la NF ISO 11290- 1. Se logró aislar Listeria monocytogenes de una muestra de pollo (2%) y de una muestra de verdura (2%), correspondiendo esta última a espárragos. Las cepas fueron aisladas empleando agar Oxford y agar Palcam como medios selectivos e identificadas mediante pruebas bioquímicas. Palabras clave: Listeria monocytogenes, pollos frescos, verduras frescas, mercados, Lima. / From the 80’s, the increase of cases of human listeriosis and their possible relationship with contaminated foods, has come worrying to the worldwide sanitary authorities. Although in our country there are not reports of an association between listeriosis and contaminated foods, but, studies have been reported that have identified this bacterium in fresh and processed marine products, raw milk and their derived. The objective of the present research was to determine the incidence of Listeria monocytogenes in chickens and vegetables, obtained of diverse centers of supply and markets of Metropolitan Lima. In total, it was analyzed 100 samples, of which 50 were of fresh chicken meat and the other 50 ones of fresh vegetables (asparagus, cabbage, celery, spinach and lettuce). The microbiological analysis was carried out according to the methodology recommended in the Bacteriological Analytical Manual of the FDA and NF ISO 11290- 1. It was achieved to isolate Listeria monocytogenes from a chicken sample (2%) and from a sample of vegetable (2%), corresponding this last one to asparaguses. The strains were isolated using Oxford agar and Palcam agar like selective mediums and identified by means of biochemical tests. Key words: Listeria monocytogenes, fresh chicken, fresh vegetables, city of Lima, markets.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2001. / Restricted until June 2002. Bibliography: leaves 105-115.

Thesis (Ph.D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xiv, 182 p.; also includes graphics. Includes abstract and vita. Advisor: Ahmed E. Yousef, Dept.of Food Science and Nutrition. Includes bibliographical references (p. ).

Listeria monocytogenes is a human and animal pathogen responsible for severe and sometimes fatal infections. Several outbreaks have been associated with contaminated commercial foodstuffs such as raw milk, soft cheese, fresh and frozen milk, poultry, seafood, fruits and vegetable products. Currently, the official method recognized by the Government of Canada for the detection and isolation of L. monocytogenes can take up to six days without confirmation, which can require two more days. An approach based on molecular beacons that fluoresce upon hybridization was developed and tested to detect L. monocytogenes and the genus Listeria in food. Two different beacons were created: one specific to species L. monocytogenes (MG1) and another for the genus Listeria (MG2). Each of these molecular beacons was used with two separate sets of primers: MG1-1f/MG1-2r, MG1-7f/MG1-4r, MG2-2f/MG2-2r and MG2-3ft/MG2-2r. (Abstract shortened by UMI.)

This study examined some of the environmental, physiological, and genetic factors or mechanisms which contribute to L. monocytogenes’ desiccation survival under food processing conditions. Desiccation experiments were carried out on stainless steel coupons stored at 43% RH, 15°C. The level of initial contamination had no impact (p>0.05), whereas the presence of a mature biofilm, prior osmoadaptation, and the presence of salt (5%) and lard (20-60%) on the SS coupons significantly (p<0.05) increased the bacterium’s desiccation survival. An Lm568 transposon mutant library was constructed to screen for novel genes involved in desiccation survival. Fifteen tolerant and 16 sensitive desiccation mutants were sequenced. Interrupted genes involved in motility and FA membrane modification were the most common in tolerant mutants whereas energy and membrane transport related genes were the most recognized in sensitive mutants. Lastly, a spontaneous desiccation resistant Lm568 variant was isolated, emphasizing the importance of understanding desiccation tolerance for food safety.

The 16S/23S ribosomal RNA (rRNA) intergenic space (IGS) regions from pathogenic and non-pathogenic species (spp.) of Listeria were characterized by the polymerase chain reaction (PCR) and DNA sequencing. DNA sequencing data for the small rRNA IGS region showed that this IGS was approximately 244 bp in length and was highly homologous (95 to 99 %) in five of the six Listeria spp examined; ie., L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, and L. ivanovii. A lower degree of homology (91 to 94 %) was detected in the large rRNA IGS region (ca. 494 bp) of these species. The DNA sequence data was used to develop two sets of oligonucleotide primers for PCR-based detection of the members of the genus Listeria. The first set of primers were Listeria genus-specific and, the second set of primers were L. monocytogenes-specific. / xv, 131 leaves ; 29 cm.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

Cryptic plasmids ranging from 2.0 to 10C kb in size were isolated from 25 out of 122 Listeria monocytogenes strains, and from 7 out of 11 strains of other Listeria species. / Of 2500 clones of a genomic library of L. monocytogenes 81-861 generated in Escherichia coli cells, 5 clones were identified in which $ beta$-hemolytic activity was stably expressed. Testing by intraperitoneal injection showed that these clones were lethal to mice. Restriction mapping of the inserts of the recombinant plasmids showed that, apart from a 650-bp internal Hind III fragment in 2 inserts, there were no other common sites. No homology was demonstrated between the DNAs of the inserts when Southern blots of restriction digests of the 5 plasmids were probed, though homology was demonstrated between the L. monocytogenes listeriolysin O gene and the DNA of one insert. The evidence suggests that at least one additional $ beta$-hemolysin, other than listeriolysin O, exists in this strain of L. monocytogenes, and that it may be a virulence factor. / Using a direct colony hybridization procedure on hydrophobic grid-membrane filters (HGMFs), the inserts of the recombinant plasmids were screened, and a DNA probe specific for L. monocytogenes was identified. After labelling with horseradish peroxidase and colour development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organism electronically. When the efficacy of the chromogen-labelled DNA probe method on HGMFs was compared with the conventional method for three artificially-inoculated foods, there were no significant differences ($ alpha$ = 0.05) shown in the recovery of L. monocytogenes from the foods.