Uma bactéria identificada como Bacillus sp. P34 isolada de intestino de peixe (Leporinus sp.) da Bacia Amazônica foi estudada quanto a sua capacidade de produzir substâncias do tipo-bacteriocina. As condições ótimas para produção da substância antimicrobiana foram determinadas. A produção da atividade antimicrobiana foi observada começando na fase exponencial de crescimento, sendo a atividade máxima observada no início da fase estacionária. Os resultados da Análise de Superfície de Resposta mostraram que a máxima produção da atividade antimicrobiana ocorreu a pH inicial entre 6.0 e 8.0 e temperaturas entre 25 e 37°C. A substância inibiu bactérias patogênicas e deteriorantes importantes em alimentos como Listeria monocytogenes, Bacillus cereus, Aeromonas hydrophila, Erwinia carotovora e Pasteurella haemolytica. O teste de termoestabilidade mostrou a perda de atividade quando a temperatura alcançou 100°C por 15 minutos. Foi sensível à ação das enzimas proteolíticas tripsina, papaína e pronase E. A substância antimicrobiana apresentou efeito bactericida e bacteriolítico sobre L. monocytogenes e B. cereus a 160 UA ml-1. O crescimento de Escherichia coli and Salmonella Enteritidis foi inibido somente quando o agente quelante EDTA foi adicionado juntamente. A atividade esporocida não foi observada. A análise da cultura de L. monocytogenes depois do tratamento com o composto antimicrobiano, usando espectroscopia de infravermelho com transformada de Fourier mostrou alterações no perfil de ácidos graxos e fosfolipídios da membrana celular bacteriana. Há evidências de que seu modo de ação interfira na membrana e na parede celular. A substância foi purificada pelo seguinte protocolo: precipitação com sulfato de amônio, cromatografias de gel filtração e de troca iônica. O peso molecular da substância foi determinado por espectroscopia de massas sendo 1498.68 Da. A substância antimicrobiana purificada apresentou sensibilidade ao tratamento com proteases e manutenção da atividade foi observada após congelamento e à incubação de 70°C por 30 minutos. / A bacterium identified as Bacillus sp. strain P34 isolated from fish intestine (Leporinus sp.) from the Amazon basin was studied in its capacity to produce bacteriocinlike substances. The optimal conditions for producing the antimicrobial activity have been established. The antimicrobial activity was produced starting at the exponencial growth phase, and maximum activity was observed at early stationary phase. Response-surface data showed that maximum antimicrobial activity production was at initial pH between 6.0 and 8.0 and temperature between 25 and 37°C. The antimicrobial substance inhibited pathogenic and spoilage food bacteria such as Listeria monocytogenes, Bacillus cereus, Aeromonas hydrophila, Erwinia carotovora and Pasteurella haemolytica. The thermoestability test showed the loss of activity when the temperature reached 100°C for 15 min. It was sensitive to the proteolytic action of trypsin, papain and pronase E. The antimicrobial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml-1. Growth of Escherichia coli and Salmonella Enteritidis was inhibited, but only when the chelating agent EDTA was co-added. Sporocidal activity was not observed. The analysis of the culture of L. monocytogenes after being treated with antimicrobial compound, using Fourier transform infrared spectroscopy, established a change in the profile that corresponding assignments of fatty acid and phospholipids. There was evidence that its mode of action to interfere with cell membrane and the cell wall. The substance was purified by the following protocol: precipitation with ammonium sulphate, gel filtration and ion exchange chromatography. The molecular weight was determined by mass spectroscopy as 1498.68 Da. Purified antimicrobial substance has shown sensitivity to protease treatment and maintained activity after freezing and incubation at 70°C for 30 min.

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. / Tesis

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.

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.