Retrospective investigation of listeriosis outbreaks in small ruminants using different analytical approaches for whole genome sequencing-based typing of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both humans and animals. While listeriosis outbreaks in humans are commonly investigated in detail, routine typing of L. monocytogenes is generally not performed in animal outbreaks. Here, seven presumable listeriosis outbreaks in small ruminants were retrospectively identified based on the pulsed-field gel electrophoresis (PFGE) profiles. Outbreaks were further characterised using three different analytical approaches based on the whole-genome sequencing (WGS) data: core-genome multilocus sequence typing (cgMLST), whole-genome MLST (wgMLST) and whole-genome single nucleotide polymorphism (wgSNP) typing. A monoclonal pattern of all seven outbreaks was identified using all three approaches, indicating common-source outbreaks. The outbreak strains belonged to sequence types (STs) 1 (n = 3), ST18 (n = 1), ST21 (n = 2) and ST184 (n = 1). Two epidemiologically linked ST1 outbreaks with indistinguishable PFGE profiles showed a polyphyletic nature and differed in >78 SNPs; thus, they were classified as separate outbreaks according to WGS. In ST184, the outbreak strain was also found in faeces of apparently healthy ruminants, silage and water collected from the trough, which were the most likely source(s) of infection. The outbreak-associated isolates differed in 0-7 cgMLST alleles, 0-12 wgMLST alleles and 1-13 SNPs. The minimum genetic diversity between outbreak-associated isolates and epidemiologically unrelated isolates of the same ST was low in all analysed cases, approaching the maximum diversity within the outbreak cluster. The results suggest that a fixed threshold to define the outbreak cluster should only be considered as a guide and highlight the role of epidemiological data for outbreak confirmation. The identified cgMLST clusters may be further investigated by wgMLST and/or wgSNP typing to increase confidence during investigations of outbreaks caused by highly clonal L. monocytogenes groups. This study gives an overview of the inter- and intra-outbreak genetic diversity of L. monocytogenes strains involved in animal outbreaks, hence improving their investigation.

Comparación de tres técnicas para subtipificación molecular de Listeria monocytogenes / Comparison of three molecular subtyping techniques for Listeria monocytogenes

Abstract Listeria monocytogenes is a foodborne pathogen. The recent alert for L. monocytogenes in vegetables from Argentina warns about the importance of reinforcing its isolation, characterization and subtyping in food, clinical and environmental samples. The aim of the present study was to compare the discriminatory power of enterobacterial repetitive interpower; genic consensus polymerase chain reaction (ERIC-PCR), automated ribotyping and pulsed-field gel electrophoresis (PFGE) to subtype strains of L. monocytogenes isolated from Argentine meat and environmental samples. Simpson's Diversity Index (DI) was calculated on the basis of based on the dendrograms obtained in the by cluster analysis, showing the following discriminatory power: ApaI-PFGE (0.980), AscI-PFGE (0.966), ribotyping (0.912), ERIC-PCR (0.886). The ID values between ApaI- and AscI-PFGE and between ribotyping and ERIC-PCR were not significantly different. Of the three techniques evaluated, PFGE showed the highest discriminatory power. However, the subtyping techniques should be accompanied by effective food monitoring strategies and reliable clinical and epidemiological studies.

Resumen Listeria monocytogenes es un patógeno alimentario. La reciente alerta por la presencia de L. monocytogenes en vegetales en Argentina advierte sobre la importancia de reforzar el aislamiento, la caracterización y la subtipificación de esta bacteria en muestras clínicas de alimentos y ambientales. El objetivo del presente estudio fue comparar el poder discriminatorio de enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), la ribotipificación automatizada y la pulsed-field gel electrophoresis (PFGE) para subtipificar cepas de L. monocytogenes aisladas de carne y de muestras ambientales en Argentina. El índice de diversidad (ID) de Simpson, calculado a partir de los dendrogramas obtenidos en el análisis de agrupamiento, mostró los siguientes resultados: Apal-PFGE (0,980), AscI-PFGE (0,966), riboti-pado (0,912), ERIC-PCR (0,886). Los valores obtenidos no fueron significativamente diferentes al comparar entre Apal- y AscI-PFGE, ni entre ribotipadoy ERIC-PCR. De las técnicas evaluadas, la PFGE presentó el mayor poder discriminatorio. Sin embargo, las técnicas de subtipificación deberían acompañarse de estrategias de control de los alimentos efectivas y de estudios clínicos y epidemiológicos confiables.

A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.

Unexpected Listeria monocytogenes detection with a dithiothreitol-based device during an aseptic hip revision.

Prosthetic joint infection diagnosis is often difficult since biofilm-embedded microorganisms attach well to the prosthetic surfaces and resist their detection by conventional methods. DL-dithiothreitol has been described as a valid method for biofilm detachment on orthopedic devices. We report the case of an occasional detection of Listeria monocytogenes in a non immuno-compromised patient with a preoperative diagnosis of aseptic loosening. The infection diagnosis due to such rare bacteria was made postoperatively, thanks to a DL-dithiothreitol-based device. This may be considered a feasible approach for the microbiological analysis of prosthetic joint infection, considering that a prompt diagnosis of such biofilm-associated infections could bring some advantages, such as an early and appropriate antibiotic therapy administration and a reduction of undiagnosed infections.

Prevalence and serotype distribution of Listeria monocytogenes isolated from foods in Montevideo-Uruguay

ABSTRACT The aim of this work was to study the prevalence of Listeria monocytogenes in foods obtained in retail shops and food industries located in Montevideo-Uruguay, and to identify the serogroups of the obtained isolates. Three-thousand one-hundred and seventy-five food samples (frozen, deli meats, ready-to-eat and cheese) were analyzed. The obtained isolates were serogrouped by multiplex PCR and serotyped by conventional procedure. Genetic comparisons were performed using pulsed-field gel electrophoresis on a sub-set of isolates belonging to the same serotype successively recovered from the same establishment. L. monocytogenes was isolated from 11.2% of samples. The highest prevalence was observed in frozen foods (38%), followed by cheese (10%). 1/2b and 4b were the most frequently identified serotypes. In six of 236 analyzed establishments we successively recovered L. monocytogenes isolates belonging to the same serotype. Most of them corresponded to serotype 1/2b. Pulsed-field gel electrophoresis profiles suggest that at least 33% of L. monocytogenes 1/2b isolates are genetically related and that may remain viable for prolonged periods. The observed prevalence of L. monocytogenes was lower than reported in neighboring countries. Our findings highlight the role that frozen foods may play in the spread of this pathogen, and the relevance of serotypes 1/2b and 4b.

Analysis of Listeria using exogenous volatile organic compound metabolites and their detection by static headspace-multi-capillary column-gas chromatography-ion mobility spectrometry (SHS-MCC-GC-IMS).

Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and D-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace-multi-capillary column-gas chromatography-ion mobility spectrometry (SHS-MCC-GC-IMS). The results obtained by SHS-MCC-GC-IMS are compared with those obtained by the more conventional analytical technique of headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity.

Listeria monocytogenes serotype 4b strains replicate in monocytes/macrophages more than the other serotypes.

We analyzed the pathogenicity of various serotypes of Listeria monocytogenes using a Balb/c mouse intravenous injection model. The survival rates of mice inoculated with strains NS1/2b (serotype 1/2b), NS3b (serotype 3b) and NS 4b (serotype 4b) were 60, 63.6 and 63.6%, respectively. Although the survival rates were similar, the bacterial growth in the liver of NS3b-infected mice was 144.5-fold higher than that in the liver of NS4b-infected mice. Histopathological analyses suggest that the NS4b strain replicated more in monocytes/macrophages, whereas the NS3b strain replicated more in hepatocytes. These results raise a possibility that the serotype 4b strains replicated more in monocytes/macrophages compared to the other serotype strains. To assess this, we isolated CD11b-positive cells from mouse livers infected with EGDe (serotype 1/2a), NS1/2b, NS3b, NS4b and the serotype 4b strains 51414 and F17 and counted the number of live bacteria in these cells. CD11b-positive cells from the NS4b-, 51414- and F17-infected mice possessed 24.4- to 42.7-fold higher numbers of live bacteria than those from mice infected with EGDe and NS3b strains. These results suggest that serotype 4b strains replicated more in monocytes/macrophages than the other serotypes, and this may be involved in the pathogenicity of serotype 4b strains, particularly in the dissemination of L. monocytogenes through the host body.

Efficacy of a Sonicating Swab for Removal and Capture of Listeria monocytogenes in Biofilms on Stainless Steel.

Listeria monocytogenes is of great concern in food processing facilities because it persists in biofilms, facilitating biotransfer. Stainless steel is commonly used for food contact surfaces and transport containers. L. monocytogenes biofilms on stainless steel served as a model system for surface sampling, to test the performance of a sonicating swab in comparison with a standard cotton swab. Swab performance and consistency were determined using total viable counts. Stainless steel coupons sampled with both types of swabs were examined using scanning electron microscopy, to visualize biofilms and surface structures (i.e., polishing grooves and scratches). Laser scanning confocal microscopy was used to image and to quantitate the biofilms remaining after sampling with each swab type. The total viable counts were significantly higher (P ≤ 0.05) with the sonicating swab than with the standard swab in each trial. The sonicating swab was more consistent in cell recovery than was the standard swab, with coefficients of variation ranging from 8.9% to 12.3% and from 7.1% to 37.6%, respectively. Scanning electron microscopic imaging showed that biofilms remained in the polished grooves of the coupons sampled with the standard swab but were noticeably absent with the sonicating swab. Percent area measurements of biofilms remaining on the stainless steel coupons showed significantly (P ≤ 0.05) less biofilm remaining when the sonicating swab was used (median, 1.1%), compared with the standard swab (median, 70.4%). The sonicating swab provided greater recovery of cells, with more consistency, than did the standard swab, and it is employs sonication, suction, and scrubbing.IMPORTANCE Inadequate surface sampling can result in foodborne illness outbreaks from biotransfer, since verification of sanitization protocols relies on surface sampling and recovery of microorganisms for detection and enumeration. Swabbing is a standard method for microbiological sampling of surfaces. Although swabbing offers portability and ease of use, there are limitations, such as high user variability and low recovery rates, which can be attributed to many different causes. This study demonstrates some benefits that a sonicating swab has over a standard swab for removal and collection of microbiological samples from a surface, to provide better verification of surface cleanliness and to help decrease the potential for biotransfer of pathogens into foods.

Clinical features and prognostic factors of listeriosis: the MONALISA national prospective cohort study.

BACKGROUND: Listeriosis is a severe foodborne infection and a notifiable disease in France. We did a nationwide prospective study to characterise its clinical features and prognostic factors. METHODS: MONALISA was a national prospective observational cohort study. We enrolled eligible cases declared to the National Reference Center for Listeria (all microbiologically proven) between Nov 3, 2009, and July 31, 2013, in the context of mandatory reporting. The outcomes were analysis of clinical features, characterisation of Listeria isolates, and determination of predictors of 3-month mortality or persisting impairment using logistic regression. A hierarchical clustering on principal components was also done for neurological and bacteraemic cases. The study is registered at ClinicalTrials.gov, number NCT01520597. FINDINGS: We enrolled 818 cases from 372 centres, including 107 maternal-neonatal infections, 427 cases of bacteraemia, and 252 cases of neurolisteriosis. Only five (5%) of 107 pregnant women had an uneventful outcome. 26 (24%) of 107 mothers experienced fetal loss, but never after 29 weeks of gestation or beyond 2 days of admission to hospital. Neurolisteriosis presented as meningoencephalitis in 212 (84%) of 252 patients; brainstem involvement was only reported in 42 (17%) of 252 patients. 3-month mortality was higher for bacteraemia than neurolisteriosis (hazard ratio [HR] 0·54 [95% CI 0·41-0·69], p<0·0001). For both bacteraemia and neurolisteriosis, the strongest mortality predictors were ongoing cancer (odds ratio [OR] 5·19 [95% CI 3·01-8·95], p<0·0001), multi-organ failure (OR 7·98 [4·32-14·72], p<0·0001), aggravation of any pre-existing organ dysfunction (OR 4·35 [2·79-6·81], p<0·0001), and monocytopenia (OR 3·70 [1·82-7·49], p=0·0003). Neurolisteriosis mortality was higher in blood-culture positive patients (OR 3·67 [1·60-8·40], p=0·002) or those receiving adjunctive dexamethasone (OR 4·58 [1·50-13·98], p=0·008). INTERPRETATION: The severity of listeriosis is higher than reported elsewhere. We found evidence of a significantly reduced survival in patients with neurolisteriosis treated with adjunctive dexamethasone, and also determined the time window for fetal losses. MONALISA provides important new data to improve management and predict outcome in listeriosis. FUNDING: Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

Rapid colorimetric sensing platform for the detection of Listeria monocytogenes foodborne pathogen.

Listeria monocytogenes is a serious cause of human foodborne infections worldwide, which needs spending billions of dollars for inspection of bacterial contamination in food every year. Therefore, there is an urgent need for rapid, in-field and cost effective detection techniques. In this study, rapid, low-cost and simple colorimetric assay was developed using magnetic nanoparticles for the detection of listeria bacteria. The protease from the listeria bacteria was detected using D-amino acid substrate. D-amino acid substrate was linked to the carboxylic acid on the magnetic nanoparticles using EDC/NHS chemistry. The cysteine residue at the C-terminal of the substrate was used for the self-assembled monolayer formation on the gold sensor surface, which in turn the black magnetic nanobeads will mask the golden color. The color will change from black to golden color upon the cleavage of the specific peptide sequence by the Listeria protease. The sensor was tested with serial dilutions of Listeria bacteria. It was found that the appearance of the gold surface area is proportional to the bacterial concentrations in CFU/ml. The lowest detection limit of the developed sensor for Listeria was found to be 2.17×10(2) colony forming unit/ml (CFU/ml). The specificity of the biosensor was tested against four different foodborne associated bacteria (Escherichia coli, Salmonella, Shigella flexnerii and Staphylococcus aureus). Finally, the sensor was tested with artificially spiked whole milk and ground meat spiked with listeria.

Rifampin phosphotransferase is an unusual antibiotic resistance kinase.

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds.

Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes.

Avaliação da atividade antimicrobiana de extratos de Eugenia anomala e Psidium salutare (Myrtaceae) frente à Escherichia coli e Listeria monocytogenes / Evaluation of the antimicrobial activity of extracts of Eugenia anomala and Psidium salutare (Myrtaceae) against the Escherichia coli and Listeria monocytogenes

RESUMO As doenças transmitidas por alimentos ocorrem principalmente devido à ingestão de alimentos contaminados por microrganismos patogênicos, dentre eles a Escherichia coli e Listeria monocytogenes. Uma das alternativas estudadas para minimizar a contaminação de alimentos é o emprego de plantas, ou seus extratos, como agentes antimicrobianos de origem natural em produtos alimentícios. Desta forma o objetivo do presente estudo é fornecer dados científicos a respeito de duas plantas nativas do RS ainda não estudadas, Eugenia anomala e Psidium salutare, visando potencial emprego como agente antimicrobiano natural em alimentos. Para tanto, avaliou-se a atividade antimicrobiana de extratos de E. anomala e P. salutare contra E. coli e L. monocytogenes através da determinação da concentração inibitória mínima (CIM) pelo método de microdiluição em caldo, a capacidade antioxidante dos extratos por meio do método de redução do radical DPPH e a citotoxicidade in vitro empregando células CHO-K1. Os resultados obtidos mostraram que os extratos de acetato de etila e etanólico de ambas as espécies possuem ação antioxidante muito alta, de 94,08% e 93,86%, respectivamente. Apenas o extrato hexânico de P. salutare apresentou ação antimicrobiana moderada (CIM = 312,5 µg/mL). Todos os extratos apresentaram ação citotóxica sendo que os maiores percentuais foram do extrato clorofórmico de E. anomala (77,05%) e hexânico de P. salutare (76,79%), na concentração de 100 µg/mL. Assim, o presente estudo demonstrou que as espécies vegetais estudadas apresentam potencial para emprego como agente antimicrobiano destes microrganismos.

ABSTRACT The foodborne diseases occur mainly due to the ingestion of food contaminated by pathogenic microorganisms, including Escherichia coli and Listeria monocytogenes. One of the alternatives studied to minimize contamination of food is the use of plants or their extracts as antimicrobial agents naturally occurring in food products. The objective of this study is to provide scientific data on two native plants of RS have not studied Eugenia anomala and Psidium salutare for a potential use as a natural antimicrobial agent in food. To this end, we evaluated the antimicrobial activity of extracts of E. anomala and P. salutare against E. coli and L. monocytogenes by determining the minimum inhibitory concentration (MIC) by the broth microdilution method, the antioxidant capacity of the extract for means DPPH radical reduction method and in vitro cytotoxicity using CHO-K1 cells. The results showed that the ethyl acetate and ethanolic extracts of both species have very high antioxidant activity, of 94.08% and 93.86%, respectively. Only the hexane extract of P. salutare showed a moderate antimicrobial activity (MIC = 312.5 mg/mL). Moreover, all extracts showed cytotoxic action of which the highest percentages were the chloroform extract of E. anomala (77.05%) and hexane P. salutare (76.79%) at a concentration of 100 mg/mL. Thus, the present study showed that plant species have potential for use as an antimicrobial agent against these microorganisms.

Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection.

The foodborne pathogen Listeria monocytogenes employs a number of virulence determinants including metalloproteases to infect hosts. Here for the first time, we identified an M29 family aminopeptidase T (encoded by lmo1603) from L. monocytogenes that possesses a typical feature to catalyze the cleavage of amino acids from peptide substrates, with a preference for arginine. The purified recombinant Lmo1603 was activated by Fe(3+), Zn(2+) and Mn(2+), but strongly stimulated by Co(2+), indicating that Lmo1603 is a cobalt-dependent aminopeptidase. Single mutation at any of the Glu216, Glu281, His308, Tyr315, His327, and Asp329 completely abolished the enzymatic activity of Lmo1603. More importantly, we showed that Lmo1603 was mainly involved in Listeria infection, but not required for growth in rich laboratory medium and minimal defined medium. Disruption of Lmo1603 resulted in almost complete attenuation of Listeria virulence in a mouse infection model. In addition, we demonstrated that Lmo1603 was mainly localized in the bacterial cytosol and required for invasion and survival inside human epithelial cells and murine macrophages. We conclude that Lmo1603 encodes a functional aminopeptidase T of M29 family, which acts as a novel intracellular virulence factor essential in the successful establishment of L. monocytogenes infections in a mouse model.

Incorporação de antimicrobianos naturais em filme biodegradável à base de alginato para o controle de Listeria monocytogenes em embutido cárneo fatiado / Incorporation of natural antimicrobials into alginate based film for the control of Listeria monocytogenes in sliced meat product

A incorporação de substâncias antimicrobianas, entre elas os óleos essenciais, em embalagens tem como objetivo minimizar a contaminação microbiana em alimentos. No entanto, essa utilização é limitada por critérios sensoriais, sendo necessário determinar a concentração mínima necessária para inibir o desenvolvimento de micro-organismos sem afetar sensorialmente as características do alimento. Assim, os objetivos dessa pesquisa foram: determinar a concentração inibitória mínima (CIM) de óleos essenciais (OE) para Listeria monocytogenes (LM); avaliar do efeito da combinação de óleos essenciais para a redução da população de LM; avaliar a combinação de compostos ativos presentes em óleos essenciais para a inibição de LM, Pseudomonas spp e Salmonella spp; avaliar in vitro e in situ a eficiência antimicrobiana do filme à base de alginato incorporado de compostos ativos presentes em óleos essenciais; determinar a disponibilidade dos compostos ativos incorporados ao filme pela determinação dos compostos fenólicos totais; caracterizar o filme frente às propriedades mecânicas e propriedades de barreira, e verificar a aceitação pelo consumidor, através da análise sensorial, de fatias de salame embaladas com o filme antimicrobiano. Constatou-se que a combinação de eugenol (0,01%) e limoneno (0,04%) apresentou um maior efeito para a redução da população de LM comparado ao uso individual desses compostos, confirmando, portanto, a existência de sinergismo entre esses dois compostos. Em relação à Pseudomonas spp, o sinergismo foi constatado quando as concentrações de eugenol (0,15%) e de limoneno (0,30%) foram utilizadas, no entanto, para Salmonella spp o mesmo efeito não foi observado. Em ensaios in vitro apesar do aumento na concentração de eugenol e limoneno incorporados à matriz do filme, não houve diferença significativa para os valores de zona de inibição antimicrobiana porém, em ensaios in situ, a utilização desse filme como embalagem primária promoveu a redução de 2 log UFC/g da população de LM até o 10º dia de análise permanecendo constante até o 30º dia o que não ocorreu com a combinação de eugenol (0,15%) e limoneno (0,3%). A quantificação dos compostos fenólicos totais nas amostras mostrou que há correlação entre o teor de compostos fenólicos extraídos da amostra do filme e o comportamento de LM. As propriedades mecânicas e de barreira do filme à base de alginato não foram significativamente afetadas pelas concentrações de eugenol e limoneno avaliadas neste estudo. O sabor, o aroma e a aparência das amostras de salame foram aceitos pelos provadores mostrando que o uso do filme à base de alginato incorporado com eugenol e limoneno é viável para a aplicação como embalagem antimicrobiana para o controle de L. monocytogenes em salame fatiado. Este resultado é considerado importante, pois em alimentos com atividade de água e valores de pH adequados à multiplicação desse patógeno e armazenados sob refrigeração, a temperatura é capaz de selecionar a microbiota presente no alimento, favorecendo a multiplicação de micro-organismos psicrotróficos, como L. monocytogenes

The incorporation of antimicrobial substances in packages aims at reducing food microbial contamination among which the phenolic compounds extract from plants have received special attention being natural and attending consumer demand. However, the use of these compounds is limited by sensory criteria and it is necessary to determine the minimum concentration required to inhibit the growth of microorganisms without affecting the sensory characteristics of the food. The aim of this research were: to determine the minimum inhibitory concentration (MIC) of essential oils (EO) for Listeria monocytogenes (LM); to evaluate effect of the combination of essential oils in order to reduce LM population; to evaluate the active compounds combination for the inhibition of LM, Pseudomonas spp, and Salmonella spp; to evaluate the antimicrobial efficiency in vitro and in situ of the alginate film incorporated with the active compounds; to determine the availability of the active compounds incorporated into the film for the determination of total phenolic compounds; to characterize the mechanical and barrier properties of the film, and to verify consumer acceptance of slices of salami packaged with the antimicrobial film. The combination of eugenol (0.01%) and limonene (0.04%) showed a greater effect for reducing LM population compared to the individual use of these compounds, confirming the existence of synergism between these two compounds. Regarding Pseudomonas spp, synergism was observed when the concentrations of eugenol (0.15%) and limonene (0.30%) were used, however, the same effect was not observed for Salmonella spp. In in vitro tests, despite of the increase in the concentration of limonene and eugenol incorporated in the film matrix, no significant difference for the antimicrobial inhibition zone values was observed. In in situ tests, the film containing eugenol (0.3%) and limonene (0.6%) promoted the reduction of 2 log CFU/g of LM population in sliced salami, while the combination of eugenol (0.15%) and limonene (0.3%) did not have the same effect. The quantification of the phenolic compounds in the film samples showed that there is correlation between the content of phenolic compounds extracted from the film sample and LM behavior. The mechanical properties of the barrier film and the alginate were not significantly affected by eugenol, and limonene concentrations evaluated in this study. The taste, flavor and appearance of the salami samples were accepted by the panel showing that the use of the alginate-based film incorporated with limonene and eugenol are feasible for use as antimicrobial packaging for the control of L. monocytogenes in sliced salami. This is an important result, because in food matrix with water activity and pH suitable for the pathogen growth, the storage under refrigerated condition is able to select the psycrothophic microorganisms, such as L. monocytogenes

Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

BACKGROUND: Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). RESULTS: All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. CONCLUSIONS: Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice model. The data indicates that MLVA complex A strains are particularly adept at establishing encephalitis possibly by virtue of their higher resistance to antibacterial defense mechanisms in microglia cells, the main target of L. monocytogenes.

Microbial species delineation using whole genome sequences.

Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail.

Adaptação e adaptação cruzada de Listeria monocytogenes aos compostos eugenol e carvacrol / Adaptation and Cross adaptation of Listeria monocytogenes to eugenol and carvacrol compounds

RESUMOA evolução de certos microrganismos permite sua rápida adaptação aos ambientes em constante mudança, desenvolvendo assim, tolerância ou resistência ao aumento de determinados estresses. O uso de compostos bioativos provenientes da flora nativa tem sido apontado como uma possível solução para os problemas de controle da resistência e proliferação bacteriana. Este trabalho visou verificar a adaptação e adaptação cruzada de L. monocytogenes, frente aos compostos fenólicos eugenol e carvacrol. A concentração mínima inibitória (CMI) dos compostos fenólicos foi determinada pela técnica de microdiluição em placas de 96 cavidades, em caldo TSB + 0,5% de Tween 80. As concentrações finais (%) obtidas foram: 0,06; 0,12; 0,24; 0,49; 0,98; 1,95; 3,9; 6,25; 12,5; 25; 50. A suspensão bacteriana padronizada foi inoculada nas cavidades das placas, as quais foram incubadas a 37°C por 24 horas com posterior leitura da absorbância a 620 nm e determinação da CMI. A adaptação das células de L. monocytogenes ao eugenol e carvacrol foi realizada com o cultivo das células em TSB + 0,06% de eugenol ou carvacrol à 37°C por 2 horas. A cultura foi então centrifugada e as células ressuspendidas e padronizadas em TSB. A seguir, realizou-se novamente a técnica de microdiluição em caldo. Os resultados obtidos demonstraram que L. monocytogenes apresentou adaptação e adaptação cruzada frente ao carvacrol e eugenol. A CMI do eugenol e carvacrol foi de 24%. A pré-exposição de L. monocutogenes a concentração sub-letal de 0,06% de carvacrol ou de eugenol aumentou sua resistência. A pré-exposição ao carvacrol promoveu a adaptação de L.monocytogenes a ele aumentando a CMI para 12,5%. Já para o eugenol a CMI passou para 25%. Quando submetidas à concentração sub-letal de eugenol, este promoveu a adaptação das células tanto ao carvacrol quanto ao eugenol, sendo a CMI de 12,5%. Os resultados obtidos demonstraram que L. monocytogenes apresentou adaptação e adaptação cruzada ao carvacrol e eugenol. O presente trabalho sugere estudos futuros ainda mais abrangentes quanto à potencialidade antimicrobiana destes compostos.

ABSTRACTSome microorganisms have evolved and therefore are able to rapidly adapt themselves to a constantly changing environment, thus developing tolerance or resistance to the increase of some specific stresses. The use of bioactive compounds from the native vegetation has been pointed out as a possible solution to the problems of control of bacterial resistance and proliferation. This work aimed to check the adaptation and cross adaptation of L. monocytogenes, towards the phenolic compounds eugenol and carvacrol. The minimum inhibitory concentration (MIC) of the phenolic compounds was determined by the microdilution technic in plates of 96 cavities, in CALDO TSB + 0,5% of Tween 80. The final concentrations (%) obtained were: 0,06; 0,12; 0,24; 0,49; 0,98; 1,95; 3,9; 6,25; 12,5; 25; 50.The patronized bacterial suspension was inoculated into the captivities of the plates, and incubated at 37°C for 24 hours with posterior reading of the absorbance at 620 nm and determination of the MIC. The adaptation of the cells of L. monocytogenes to the eugenol and carvacrol was made through the cultivation of the cells in TSB + 0,06% of eugenol or carvacrol at 37°C for 2 hours. The sample was then centrifuged and the cells were re-suspended and patronized in TSB. After that, the microdilution technic was performed one more time. The obtained results revealed that the L. monocytogenespresented adaptation and cross adaptation to the eugenol and carvacrol. The eugenol and carvacrol CMI was at 24%. The pre-exposition of L. monocytogenes to sub-lethal doses of 0,06% of eugenol or carvacrol enhanced its resistance. The pre-exposition to carvacrol promoted the adaptation of L. monocytogenes to it thus increasingthe MIC to 12,5%. To the eugenol the CMI got to 25%. When submitted to sub-lethal concentrations of eugenol, the latterpromoted the adaptation of the cells to carvacrol and eugenol, bringing the CMI to 12,5%. The obtained results showed that theL. monocytogenespresented adaptation and cross adaptation to the eugenol and carvacrol. The current work suggests future studies even broader regarding the antimicrobial potentialityof these compounds.