Evaluation of Antimicrobial Activity by Marine Nocardiopsis dassonvillei against Foodborne Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli.

The emergence of multidrug-resistant pathogens creates public health challenges, prompting a continuous search for effective novel antimicrobials. This study aimed to isolate marine actinomycetes from South Africa, evaluate their in vitro antimicrobial activity against Listeria monocytogenes and Shiga toxin-producing Escherichia coli, and characterize their mechanisms of action. Marine actinomycetes were isolated and identified by 16S rRNA sequencing. Gas chromatography-mass spectrometry (GC-MS) was used to identify the chemical constituents of bioactive actinomycetes' secondary metabolites. Antibacterial activity of the secondary metabolites was assessed by the broth microdilution method, and their mode of actions were predicted using computational docking. While five strains showed antibacterial activity during primary screening, only Nocardiopsis dassonvillei strain SOD(B)ST2SA2 exhibited activity during secondary screening for antibacterial activity. GC-MS identified five major bioactive compounds: 1-octadecene, diethyl phthalate, pentadecanoic acid, 6-octadecenoic acid, and trifluoroacetoxy hexadecane. SOD(B)ST2SA2's extract demonstrated minimum inhibitory concentration and minimum bactericidal concentration, ranging from 0.78-25 mg/mL and 3.13 to > 25 mg/mL, respectively. Diethyl phthalate displayed the lowest bacterial protein-binding energies (kcal/mol): -7.2, dihydrofolate reductase; -6.0, DNA gyrase B; and -5.8, D-alanine:D-alanine ligase. Thus, marine N. dassonvillei SOD(B)ST2SA2 is a potentially good source of antibacterial compounds that can be used to control STEC and Listeria monocytogenes.

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions.

BACKGROUND: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. RESULTS: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L-1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. CONCLUSIONS: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.

Human Listeriosis Presenting as Breast Abscess: Report of a Rare Case.

An abscess is defined as a collection of pus in various tissues of the body including skin and other organs. Abscesses most commonly are formed on the skin under the armpits, groin areas, and rectal areas. Most abscesses involve microbial infections with few remaining sterile. The treatment of abscesses includes both medical and surgical intervention. In the era of multidrug resistance, isolation and identification of the causative microbe and testing for antimicrobial susceptible patterns assume greater significance for the better management of patients, thereby reducing the resultant morbidity and mortality. Listeria spp. are a group of aerobic and non-spore forming gram-positive bacilli. They are present in the environment, soil, and water. Listeria spp. have also been noted to be present as a normal intestinal flora of animals. They are known for their ability to thrive under both cold and hot environmental conditions. Human infections with Listeria spp. have not been frequently reported, mostly because of the difficulty in laboratory identification and complex clinical presentations. In humans, Listeria spp. have been frequently responsible for food poisoning and neonatal meningitis. Although not considered as a classic pathogen, Listeria spp. are associated with infections in elderly people, pregnant women, newborns, and persons with weakened immune systems. This report presents a case of breast abscess caused by Listeria spp. in a young lactating female belonging to rural India.

Assessment of virulence factors, antibiotic resistance and amino-decarboxylase activity in Enterococcus faecium MXVK29 isolated from Mexican chorizo.

Enterococcus faecium MXVK29 has the ability to produce an antimicrobial compound that belongs to Class IIa of the Klaenhammer classification, and could be used as part of a biopreservation technology through direct inoculation of the strain as a starter or protective culture. However, Enterococcus is considered as an opportunistic pathogen, hence, the purpose of this work was to study the food safety determinants of E. faecium MXVK29. The strain was sensitive to all of the antibiotics tested (penicillin, tetracycline, vancomycin, erythromycin, chloramphenicol, gentamicin, neomycin, kanamycin and netilmicin) and did not demonstrate histamine, cadaverine or putrescine formation. Furthermore, tyrosine-decarboxylase activity was detected by qualitative assays and PCR. Among the virulence factors analysed for the strain, only the genes encoding the sexual pheromone cCF10 precursor lipoprotein (ccf) and cell-wall adhesion (efaAfm ) were amplified. The presence of these genes has low impact on pathogenesis, as there are no other genes encoding for virulence factors, such as aggregation proteins. Therefore, Enterococcus faecium could be employed as part of a bioconservation method, because it does not produce risk factors for consumer's health; in addition, it could be used as part of the hurdle technology in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of molecular techniques has allowed, in recent years, to detect pathogenicity genes present in the genome of starter cultures used in food processing and preservation. The presence of these genes is undesirable, because horizontal transfer may occur with the natural biota of consumers. For this reason, it is important to analyse the presence of pathogenicity genes in such cultures. In this work, virulence factors and antibiotic resistance of Enterococcus faecium strain MXVK29, producing an antimicrobial compound with high antilisterial activity, were analysed. The results indicate that the strain is safe to be used in food processing as starter culture.

Effect of Filling Type and Heating Method on Prevalence of Listeria species and Listeria monocytogenes in Dumplings Produced in Poland.

The count of Listeria monocytogenes was determined, before and after heat treatment, in 200 samples of dumplings of 9 brands and with different types of stuffing. Analyses were conducted according to ISO 11290-1 standard and with real-time PCR method. The highest count of L. monocytogenes was found in meat dumplings (10(2) to 10(4) CFU/g), whereas products with white cheese-potato stuffing and vegetable-mushroom stuffing contained significantly less Listeria, 20 to 80 and 5 to 32 CFU/g, respectively. In cooled meat dumplings the extent of contamination depended significantly on the producer. In addition, a significant (P < 0.05) correlation was determined between contamination level and meat content in the stuffing (rho = 0.418), especially in stuffing containing pork meat (0.464), contrary to beef-containing stuffing (0.284). Heating dumplings in boiling water for 2 min completely eliminated L. monocytogenes in meat dumplings. In contrast, the microwave heating applied for 2 min at 600 W only reduced the count of L. monocytogenes by 1 to 2 logs. Hence, the microwave heating failed to reduce the risk of infection with this pathogen below the level permissible in the EU regulation, especially in the most contaminated samples. In this case, the efficacy of microwave heating was significantly (P < 0.05) affected by the initial count of L. monocytogenes (rho = 0.626), then by meat content in the stuffing (0.476), and to the lowest extent--by the type of meat (0.415 to 0.425). However, no Listeria sp. and L. monocytogenes were isolated from cooked dumplings with fruits (strawberries or blueberries).

Enzyme-linked imunoassays for the detection of Listeria sp. and Salmonella sp. in sausage: a comparison with conventional methods

This study was carried out comparing the conventional methods (ISO 11290-1 and BAM method, 2008) and system mini-Vidas® (Biomerieux), for detection of Listeria sp. and Salmonella sp. in cooled sausage. The immunoenzymatic method has shown to be effective for the detection of target pathogens, it has presented itself as an excellent screening method.

Enzyme-linked imunoassays for the detection of Listeria sp. and Salmonella sp. in sausage: a comparison with conventional methods.

This study was carried out comparing the conventional methods (ISO 11290-1 and BAM method, 2008) and system mini-Vidas® (Biomerieux), for detection of Listeria sp. and Salmonella sp. in cooled sausage. The immunoenzymatic method has shown to be effective for the detection of target pathogens, it has presented itself as an excellent screening method.

Knowledge Mapping for Climate Change and Food- and Waterborne Diseases.

The authors extracted from the PubMed and ScienceDirect bibliographic databases all articles published between 1998 and 2009 that were relevant to climate change and food- and waterborne diseases. Any material within each article that provided information about a relevant pathogen and its relationship with climate and climate change was summarized as a key fact, entered into a relational knowledge base, and tagged with the terminology (predefined terms) used in the field. These terms were organized, quantified, and mapped according to predefined hierarchical categories. For noncholera Vibrio sp. and Cryptosporidium sp., data on climatic and environmental influences (52% and 49% of the total number of key facts, respectively) pertained to specific weather phenomena (as opposed to climate change phenomena) and environmental determinants, whereas information on the potential effects of food-related determinants that might be related to climate or climate change were virtually absent. This proportion was lower for the other pathogens studied (Campylobacter sp. 40%, Salmonella sp. 27%, Norovirus 25%, Listeria sp. 8%), but they all displayed a distinct concentration of information on general food-and water-related determinants or effects, albeit with little detail. Almost no information was available concerning the potential effects of changes in climatic variables on the pathogens evaluated, such as changes in air or water temperature, precipitation, humidity, UV radiation, wind, cloud coverage, sunshine hours, or seasonality. Frequency profiles revealed an abundance of data on weather and food-specific determinants, but also exposed extensive data deficiencies, particularly with regard to the potential effects of climate change on the pathogens evaluated. A reprioritization of public health research is warranted to ensure that funding is dedicated to explicitly studying the effects of changes in climate variables on food- and waterborne diseases.

Climate Change Impact Assessment of Food- and Waterborne Diseases.

The PubMed and ScienceDirect bibliographic databases were searched for the period of 1998-2009 to evaluate the impact of climatic and environmental determinants on food- and waterborne diseases. The authors assessed 1,642 short and concise sentences (key facts), which were extracted from 722 relevant articles and stored in a climate change knowledge base. Key facts pertaining to temperature, precipitation, water, and food for 6 selected pathogens were scrutinized, evaluated, and compiled according to exposure pathways. These key facts (corresponding to approximately 50,000 words) were mapped to 275 terminology terms identified in the literature, which generated 6,341 connections. These relationships were plotted on semantic network maps to examine the interconnections between variables. The risk of campylobacteriosis is associated with mean weekly temperatures, although this link is shown more strongly in the literature relating to salmonellosis. Irregular and severe rain events are associated with Cryptosporidium sp. outbreaks, while noncholera Vibrio sp. displays increased growth rates in coastal waters during hot summers. In contrast, for Norovirus and Listeria sp. the association with climatic variables was relatively weak, but much stronger for food determinants. Electronic data mining to assess the impact of climate change on food- and waterborne diseases assured a methodical appraisal of the field. This climate change knowledge base can support national climate change vulnerability, impact, and adaptation assessments and facilitate the management of future threats from infectious diseases. In the light of diminishing resources for public health this approach can help balance different climate change adaptation options.

Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars) with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA) and CM 1084). The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite differences in composition of the chromogenic media assessed, these disparities did not affect concordance among results. However, PALCAM agar was shown to suppress other microorganisms more effectively, being more applicable for detecting Listeria strains present in lower quantities. Based on these results, the use of PALCAM agar, in combination with a chromogenic media, is recommended for enhanced isolation of atypical Listeria sp. strains in meat products.

Este estudo teve como objetivo a análise da prevalência de Listeria sp. em linguiças resfriadas e a comparação dos meios seletivos utilizados no plaqueamento do método ISO 11290-1 (Ágar PALCAM e Ágar Oxford), e ágares cromogênicos (Ágares Listeria Cromogênico CM 1080 (OCLA) e CM 1084 (ISO)). A frequência de Listeria sp. foi de 52,9%, sendo que destas, 13,7% corresponderam à L. monocytogenes. A eficácia dos quatro ágares para o isolamento de L. monocytogenes demonstrou-se satisfatória. Apesar de haver algumas diferenças nas composições dos meios cromogênicos analisados, estas não pareceram influenciar nas concordâncias entre os resultados expressos. Contudo, o ágar PALCAM mostrou-se mais eficaz na supressão de outros micro-organismos, aumentando, assim, a possibilidade de detecção de espécies de Listeria presentes em número reduzido. Através deste trabalho sugere-se a utilização do ágar PALCAM associado a um meio cromogênico para aumentar a chance de isolamento de cepas atípicas de Listeria sp. em produtos cárneos.

Salmonella sp e Listeria monocytogenes em presunto suíno comercializado em supermercados de Fortaleza (CE, Brasil): fator de risco para a saúde pública / Salmonella sp and Listeria monocytogenes in fully cooked ham commercialized in supermarkets of Fortaleza (CE, Brazil): risk factor for public health

O objetivo deste trabalho foi avaliar a ocorrência de Salmonella sp. e Listeria spp. em presunto suíno cozido sem capa de gordura, mantido sob temperatura de refrigeração, comercializado em supermercados de Fortaleza (CE). O material estudado compreendeu quarenta amostras, provenientes de oito marcas comerciais, coletadas em 26 estabelecimentos. A pesquisa de Salmonella sp. e Listeria spp. foi efetuada segundo APHA e HBP, respectivamente. Foi constatado que 30 por cento das amostras estavam contaminadas por Salmonella sp. e 42,50 por cento, 22,50 por cento e 2,5 por cento por L. monocytogenes, L. innocua e L. welshimeri, respectivamente. Foram observadas, ainda, falhas na aplicação das boas práticas de manipulação na área de frios dos supermercados. Verificou-se que 34,61 por cento, 57,69 por cento e 15,38 por cento dos estabelecimentos apresentaram alguma amostra contaminada por Salmonella sp., L. monocytogenes e ambos os microrganismos, respectivamente, abrangendo a área de fiscalização das seis Secretarias Executivas Regionais (SER) do município de Fortaleza. Estes dados mostram a necessidade de um aperfeiçoamento quanto ao controle de qualidade em todo elo da cadeia alimentar como fator fundamental na prevenção de enfermidades transmitidas por alimentos (ETA).

The objective of this work was to evaluate the occurrence of Salmonella sp. and Listeria spp. in fully cooked ham without fat cover, stored under refrigeration, commercialized in supermarkets of Fortaleza (CE, Brazil). Forty samples of cooked ham, proceeding from eight trademarks, collected in 26 establishments, were analyzed according to the APHA and HBP for Salmonella sp. and Listeria spp. detection, respectively. Thirty percent of the samples were contaminated with Salmonella sp. and 42.50 percent, 22.50 percent e 2.5 percent with L. monocytogenes, L. innocua e L. welshimeri, respectively. It was also observed imperfections in the application of good manipulation practices in the supermarkets freezer section. It was verify that 34.61 percent, 57.69 percent e 15.38 percent of the establishments presented some sample contaminated with Salmonella sp., L. monocytogenes and both microorganisms respectively, including the area inspected by the six Regionals Executive General Offices of Fortaleza. These data show the necessity of a quality control improvement in all parts of food systems as an essential factor in the prevention of foodborne diseases.

Meningoencefalite por Listeria monocytogenes em ovinos / Meningoencephalitis in sheep caused by Listeria monocytogenes

São descritos sete casos de doença neurológica em ovinos por Listeria monocytogenes no Rio Grande do Sul e Paraná entre 2000 e 2007. Foram afetados ovinos com idades entre 12-24 meses. Os casos ocorreram no verão e início da primavera e os índices gerais de morbidade e letalidade foram de 3,15 por cento e 100 por cento, respectivamente. Quando essa informação estava disponível, nenhum dos ovinos afetados era alimentado com silagem. Em três propriedades havia contato próximo dos ovinos afetados com outras espécies. A evolução do quadro clínico foi de 12 horas a três dias e os sinais clínicos foram caracterizados por decúbito (7/7), desvio da cabeça (4/7), incoordenação (3/7), depressão (3/7), andar em círculos (2/7), cegueira unilateral, emagrecimento progressivo, febre, midríase, movimentos de pedalagem, nistagmo lateral, opistótono, paralisia flácida dos membros pélvicos ou dos quatro membros, salivação excessiva e tremores (1/7 cada). Histologicamente observou-se encefalite com microabscessos, predominantemente unilateral com variáveis graus de gliose e alterações degenerativas como esferóides axonais e infiltração de células Gitter. As lesões se estendiam desde a medula oblonga até o mesencéfalo. Antígenos de Listeria monocytogenes foram detectados por imuno-histoquímica em seções de tronco encefálico de todos os ovinos afetados. O diagnóstico foi realizado com base nos achados epidemiológicos e clinico-patológicos, e confirmado pela imuno-histoquímica (IHQ) utilizando anticorpo policlonal anti-L. monocytogenes.

Seven cases of neurological disease in sheep caused by Listeria monocytogenes in Rio Grande do Sul and Paraná state, southern Brazil are described. The cases occurred between 2000 and 2007 and 12-24-month-old sheep were affected. Overall morbidity and lethality rates were 3.15 percent and 100 percent, respectively. Cases occurred in the summer and early spring. When this information was available, affected sheep had not been fed with silage. In three farms there were close contact among affected sheep and other species. Clinical signs were characterized by recumbency (7/7), head tilt (4/7), incoordination (3/7), depression (3/7), circling (2/7), unilateral blindness, wasting, fever, midriasis, paddling, opisthotonus, hind or hind and fore limb paralysis, drooling, and muscle tremors (1/7 each). Clinical evolution varied from 12 hours to three days. Histological findings consisted of predominantly unilateral, microabscedative encephalitis with variable degrees of gliosis and degenerative lesions characterized by axonal spheroids and infiltration by Gitter cells. These lesions were observed extending from medulla oblongata to mesencephalon. Listeria monocytogenes antigen was showed by imunohistochemistry in routinely processed sections of brainstem from all seven affected sheep. The diagnostic was based on epidemiological, clinical, and pathological findings and confirmed by immunohistochemistry (IHQ) using polyclonal anti-L. monocytogenes antibody.

Ocorrência de Listeria monocytogenes em queijo artesanal tipo coalho comercializado na cidade de Manaus-AM, Brasil / Occurrence of Listeria monocytogenes in handmade type cheese coagulum marketed in the city of Manaus-Amazonas, Brazil

A ocorrência de Listeria sp. em queijo artesanal tipo coalho comercializado na cidade de Manaus foi estudada. No período de março a maio de 1998 foram coletadas 58 amostras do produto em feiras e mercados localizados em seis zonas da cidade de Manaus. Em duas amostras de queijo foram identificadas duas espécies de Listeria: L. monocytogenes e L. innocua. A primeira espécie é reconhecidamente patogênica para o ser humano. Os resultados apresentados demonstraram haver risco de transmissão de enfermidades pelo consumo de queijos processados sem os devidos cuidados de higiene.

The occurrence of Listeria sp. in cheese craft type coagulum marketed in the city of Manaus was studied. In the period of March to May of 1998 58 samples of the product were collected at fairs and located markets in six areas of the city. In two cheese samples we identified two species of Listeria: L. monocytogenes and L. innocua. The first species was found fobe pathogenic for the human being. The results demonstrated there to be risk of transmission of illnesses from the consumption of cheeses processed without the due hygiene care.