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1.
Int J Food Microbiol ; 350: 109247, 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34023680

RESUMO

The ability of Listeria monocytogenes isolates to survive within the food production environment (FPE), as well as virulence, varies greatly between strains. There are specific genetic determinants that have been identified which can strongly influence a strains ability to survive in the FPE and/or within human hosts. In this study, we assessed the FPE fitness and virulence potential, including efficacy of selected hygiene or treatment intervention, against 52 L. monocytogenes strains isolated from various food and food environment sources. Phenotypic tests were performed to determine the minimum inhibitory concentration of cadmium chloride and benzalkonium chloride and the sensitivities to five clinically relevant antibiotics. A genomic analysis was also performed to identify resistance genes correlating to the observed phenotypic resistance profiles, along with genetic determinants of interest which may elude to the FPE fitness and virulence potential. A transposon element containing a novel cadmium resistance gene, cadA7, a Tn916 variant insert in the hypervariable Listeria genomic island 1 region and an LGI2 variant were identified. Resistance to cadmium and disinfectants was prevalent among isolates in this study, although no resistance to clinically important antimicrobials was observed. Potential hypervirulent strains containing full length inlA, LIPI-1 and LIPI-3 were also identified in this study. Cumulatively, the results of this study show a vast array of FPE survival and pathogenicity potential among food production-associated isolates, which may be of concern for food processing operators and clinicians regarding L. monocytogenes strains colonising and persisting within the FPE, and subsequently contaminating food products then causing disease in at risk population groups.


Assuntos
Antibacterianos/farmacologia , Compostos de Benzalcônio/farmacologia , Cloreto de Cádmio/farmacologia , Desinfetantes/farmacologia , Farmacorresistência Bacteriana/genética , Listeria monocytogenes/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Manipulação de Alimentos , Microbiologia de Alimentos , Ilhas Genômicas/genética , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Testes de Sensibilidade Microbiana , Virulência/genética , Fatores de Virulência/genética
2.
Nat Commun ; 12(1): 1704, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731716

RESUMO

GPR37 was discovered more than two decades ago, but its biological functions remain poorly understood. Here we report a protective role of GPR37 in multiple models of infection and sepsis. Mice lacking Gpr37 exhibited increased death and/or hypothermia following challenge by lipopolysaccharide (LPS), Listeria bacteria, and the mouse malaria parasite Plasmodium berghei. Sepsis induced by LPS and Listeria in wild-type mice is protected by artesunate (ARU) and neuroprotectin D1 (NPD1), but the protective actions of these agents are lost in Gpr37-/- mice. Notably, we found that ARU binds to GPR37 in macrophages and promotes phagocytosis and clearance of pathogens. Moreover, ablation of macrophages potentiated infection, sepsis, and their sequelae, whereas adoptive transfer of NPD1- or ARU-primed macrophages reduced infection, sepsis, and pain-like behaviors. Our findings reveal physiological actions of ARU in host cells by activating macrophages and suggest that GPR37 agonists may help to treat sepsis, bacterial infections, and malaria.


Assuntos
Macrófagos/metabolismo , Dor/prevenção & controle , Receptores Acoplados a Proteínas G/metabolismo , Sepse/prevenção & controle , Transferência Adotiva , Animais , Artesunato/metabolismo , Artesunato/farmacologia , Artesunato/uso terapêutico , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Lipopolissacarídeos/toxicidade , Listeria monocytogenes/patogenicidade , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Macrófagos/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Dor/imunologia , Dor/mortalidade , Fagocitose/efeitos dos fármacos , Plasmodium berghei/patogenicidade , Receptores Acoplados a Proteínas G/deficiência , Sepse/imunologia , Sepse/mortalidade , Sepse/terapia
3.
Toxicol Appl Pharmacol ; 415: 115441, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33556388

RESUMO

The immunotoxicity of zearalenone (ZEA) and deoxynivalenol (DON), two of the most common environmental mycotoxins, has been well investigated. However, due to the complexity of the immune system, especially during bacterial infection, many types of immune cells are involved in invasion resistance and bacterial clearance. Of these, T helper 2 (Th2) cells, which are members of the helper T cell family, assist B cells to activate and differentiate into antibody-secreting cells, participate in humoral immune response, and, ultimately, eliminate pathogens. Thus, it is important to identify the stage at which these toxins affect the immune function, and to clarity the underlying mechanisms. In this study, mice infected with Listeria monocytogenes (Listeria) were used to study the effects of ZEA, DON, and ZEA + DON on Th2 differentiation, Interleukin-4 Receptor (IL-4R) expression, costimulatory molecules expression and cytokine secretion after Listeria infection. Naive CD4+ T cells, isolated from mice, were used to verify the in vivo effects and the associated mechanisms. In vivo experiments showed that these toxins aggravated spleen damage after Listeria infection and reduced the differentiation of Th2 cells by affecting the synthesis of IL-4R of CD4+ T cells. In addition, the level of the costimulatory molecule CD154 decreased. Consistent with this, in vitro studies showed that these toxins inhibited the differentiation of mouse naive CD4+ T cell into Th2 subtype and decreased IL-4R levels. In addition, the levels of costimulatory molecules CD154, CD278 and the Th2 cells secrete cytokines IL-4, IL-6, and IL-10 decreased. Based on our in vivo and in vitro experiments, we suggest that ZEA, DON, and ZEA + DON inhibit the expression of costimulatory molecules on CD4+ T cell, and inhibit the IL-4R-mediated Th2 cell differentiation. This may indicate that the body cannot normally resist or clear the pathogen after mycotoxin poisoning.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Listeriose/induzido quimicamente , Ativação Linfocitária/efeitos dos fármacos , Receptores de Interleucina-4/metabolismo , Baço/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Tricotecenos/toxicidade , Zearalenona/toxicidade , Animais , Ligante de CD40/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/metabolismo , Listeriose/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/microbiologia
4.
Nature ; 590(7846): 457-462, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33568812

RESUMO

In contrast to nearly all other tissues, the anatomy of cell differentiation in the bone marrow remains unknown. This is owing to a lack of strategies for examining myelopoiesis-the differentiation of myeloid progenitors into a large variety of innate immune cells-in situ in the bone marrow. Such strategies are required to understand differentiation and lineage-commitment decisions, and to define how spatial organizing cues inform tissue function. Here we develop approaches for imaging myelopoiesis in mice, and generate atlases showing the differentiation of granulocytes, monocytes and dendritic cells. The generation of granulocytes and dendritic cells-monocytes localizes to different blood-vessel structures known as sinusoids, and displays lineage-specific spatial and clonal architectures. Acute systemic infection with Listeria monocytogenes induces lineage-specific progenitor clusters to undergo increased self-renewal of progenitors, but the different lineages remain spatially separated. Monocyte-dendritic cell progenitors (MDPs) map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1. Specific deletion of Csf1 in endothelium disrupts the architecture around MDPs and their localization to sinusoids. Subsequently, there are fewer MDPs and their ability to differentiate is reduced, leading to a loss of nonclassical monocytes and dendritic cells during both homeostasis and infection. These data indicate that local cues produced by distinct blood vessels are responsible for the spatial organization of definitive blood cell differentiation.


Assuntos
Rastreamento de Células/métodos , Células Mieloides/citologia , Mielopoese , Coloração e Rotulagem/métodos , Animais , Atlas como Assunto , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Linhagem da Célula , Autorrenovação Celular , Células Dendríticas/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Granulócitos/citologia , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Fator Estimulador de Colônias de Macrófagos/deficiência , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Monócitos/citologia , Células Mieloides/metabolismo
5.
Trop Anim Health Prod ; 53(1): 127, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33454847

RESUMO

The frequency of Listeria monocytogenes isolates collected from a total of 1150 samples including food (n = 300), livestock (n = 50), and human clinical (n = 800) was evaluated during 2008-2016. Antimicrobial resistance patterns, virulence factors, and molecular characteristics of these isolates were analyzed using disk diffusion method, sequencing, serotyping, and pulsed-field gel electrophoresis (PFGE). The analysis of 44 L. monocytogenes isolates showed that 72.7% (32 of 44) of all the isolates belonged to Serotype 1/2c, and 15.9% (7 of 44) belonged to Serotype 3c. All 44 isolates were resistant to one or more antimicrobial agents with the most frequent resistance to penicillin (75%) and tetracycline (47.7%). Of the 44 L. monocytogenes strains, 100, 69.2, and 62.5% of livestock, human, and food strains were resistant to penicillin, respectively. Using pulsed-field gel electrophoresis (PFGE) technique, the isolates' genetic diversity was determined, and 28 PFGE patterns with 8 common (CT) and 20 single types (ST) were identified. This study highlights the high prevalence of Serotype 1/2c in clinical and livestock samples, while different serotypes were observed in food samples. The presence of rare serotypes such as 4c, belonging to the Lineage III, as well as 4e and 1/2c which are infrequent in Iran indicates that paying attention to uncommon serotypes, especially 1/2c, during the listeriosis outbreaks is necessary.


Assuntos
Microbiologia de Alimentos , Listeria monocytogenes , Listeriose , Virulência , Animais , Técnicas de Tipagem Bacteriana , Farmacorresistência Bacteriana/genética , Eletroforese em Gel de Campo Pulsado , Humanos , Irã (Geográfico)/epidemiologia , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose/epidemiologia , Listeriose/veterinária , Gado/microbiologia , Tipagem Molecular , Sorotipagem
6.
Int J Food Microbiol ; 340: 109043, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33454520

RESUMO

The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed for the presence of Listeria monocytogenes. A total of 18 L. monocytogenes isolates were obtained from 229 samples, and their genomes were sequenced to perform comparative genomic analyses. An increase in the frequency of isolation of L. monocytogenes and in the diversity of sequence types (STs) detected was observed along time. Although the strains isolated belonged to six different STs (ST8, ST9, ST14, ST37, ST121 and ST155), ST9 was the most abundant (8 out of 18 strains). Low (0 and 2) single nucleotide polymorphism (SNP) distances were found between two pairs of ST9 strains isolated in both cases 3 months apart from the same processing room (Lm-1267 and Lm-1705, with a 2 SNPs distance in the core genome; Lm-1265 and Lm-1706, with a 0 SNPs distance), which suggests that these strains may be persistent L. monocytogenes strains in the food processing environment. Most strains showed an in silico attenuated virulence potential either through the truncation of InlA (in 67% of the isolates) or the absence of other virulence factors involved in cell adhesion or invasion. Twelve of the eighteen L. monocytogenes isolates contained a plasmid, which ranged in size from 4 to 87 Kb and harbored stress survival, in addition to heavy metals and biocides resistance determinants. Identical or highly similar plasmids were identified for various sets of L. monocytogenes ST9 isolates, which suggests the clonal expansion and persistence of plasmid-containing ST9 strains in the processing environments of the meat facility. Finally, the analysis of the L. monocytogenes genomes available in the NCBI database, and their associated metadata, evidenced that strains from ST9 are more frequently reported in Europe, linked to foods, particularly to meat and pork products, and less represented among clinical isolates than other L. monocytogenes STs. It also showed that the ST9 strains here isolated were more closely related to the European isolates, which clustered together and separated from ST9 North American isolates.


Assuntos
Contaminação de Equipamentos , Manipulação de Alimentos , Variação Genética , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Instalações Industriais e de Manufatura , Carne , Animais , Desinfetantes , Europa (Continente) , Pisos e Cobertura de Pisos , Microbiologia de Alimentos , Genes Bacterianos , Listeria monocytogenes/classificação , Listeria monocytogenes/patogenicidade , Plasmídeos , Suínos , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
7.
Int J Food Microbiol ; 340: 109057, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33460999

RESUMO

Various adverse conditions can trigger defensive mechanisms in Listeria monocytogenes that can increase the virulence of surviving cells. The objective of this study was to evaluate the expression of one stress-response (sigB) and three virulence (plcA, hly, and iap) genes in L. monocytogenes exposed to a sub lethal dose of E-beam irradiation in dry-cured ham. To accomplish this, dry-cured ham slices (10 g) were immersed in a 109 CFU/mL suspension of L. monocytogenes strain S4-2 and subsequently irradiated with 1, 2, or 3 kGy. After irradiation, samples were stored at 7 °C or 15 °C for 30 days. Absolute gene expression levels were determined by RT-qPCR, and numbers of surviving Listeria cells were assessed by microbial counts after different storage times (0, 7, 15, and 30 days). At 7 °C, after E-beam treatment at doses of 2 or 3 kGy, Listeria gene expression significantly increased (p ≤ 0.05) up to day 15. Listeria counts decreased with increasing dosage. The relationship between absolute gene expression and the number of surviving Listeria cells could indicate that sublethal doses of E-beam irradiation can increase expression of the genes studied. We observed no significant influence of storage time or temperature on gene expression (p > 0.05). Listeria that survives E-beam treatment may display increased virulence, constituting a significant potential public health risk.


Assuntos
Irradiação de Alimentos , Listeria monocytogenes/genética , Listeria monocytogenes/efeitos da radiação , Carne de Porco/microbiologia , Animais , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Conservação de Alimentos , Expressão Gênica , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Fator sigma/genética , Estresse Fisiológico/genética , Suínos , Temperatura , Virulência/genética
8.
Int J Food Microbiol ; 339: 109023, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33341686

RESUMO

The aim of the present study is to investigate the prevalence and genetic diversity of Listeria monocytogenes in various fresh and frozen vegetable products available in Poland. The samples were collected at retail market within the framework of national official control and monitoring program. In the years 2016-2019 a total of 49 samples out of 8712 collected vegetable samples were positive for L. monocytogenes. Our findings demonstrated that the occurrence of L. monocytogenes in various vegetable products was generally low, on average only 0.56% in the studied years. All isolates were susceptible to 11 antimicrobial agents: penicillin, ampicillin, meropenem, erythromycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, ciprofloxacin, chloramphenicol, gentamicin, vancomycin, and tetracycline. All of them harbored virulence-associated genes (inlA, inlC, and lmo2672), 82% harbored inlJ gene and few of them (22%) also possessed the llsX gene. The majority of collected isolates (65%) belonged to molecular serogroup 1/2a-3a, followed by 4ab-4b-4d-4e (33%), and only one to serogroup 1/2b-3b-7 (2%). Isolates yielded 18 different restriction profiles, revealing a large cluster of contamination linked to frozen corn (21 strains) and distributed in 3 pulsotypes. MLST analysis classified selected isolates into nine clonal complexes (CCs). The obtained results contribute to characterizing the diversity of L. monocytogenes isolated from various vegetable products in Poland and their impact on food safety and public health.


Assuntos
Microbiologia de Alimentos , Variação Genética , Listeria monocytogenes/genética , Verduras/microbiologia , Antibacterianos/farmacologia , Microbiologia de Alimentos/estatística & dados numéricos , Humanos , Incidência , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Tipagem de Sequências Multilocus , Polônia , Sorogrupo , Sorotipagem , Virulência/genética
9.
Protein Expr Purif ; 177: 105760, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002609

RESUMO

Resistance to antibiotics is a serious concern to treat infectious diseases and also, for food preservation. Existing antibiotics generally inhibit enzymes participating in key bacterial processes, such as formation of cell wall, replication, transcription and translation. However, bacteria have rapidly evolved new mechanisms to combat these antibiotics and it hence becomes indispensable to identify newer targets and identify/design inhibitors against them. Another concern is that most antibiotics are broad spectrum; they largely bind and inhibit the active site of the target enzyme. Rel proteins, which synthesize (and hydrolyze) (p)ppGpp in response to a variety of stress encountered by bacteria, is a profitable target owing to its distinct absence in humans and an intricate regulation of the catalytic activities. Inactivation of (p)ppGpp synthesis by Rel, disables bacterial survival in Mycobacterium tuberculosis and Staphylococcus aureus, while inactivating the hydrolysis activity was lethal. The poor MIC values of the currently known Rel inhibitors present a distinct opportunity to develop better inhibitors and warrants a detailed structural characterization and understanding of the complex regulation in Rel proteins. It will open new avenues for the design of effective, species-specific inhibitors. In an attempt to identify unique sites for inhibitor design using structure-based approaches, we initiate a study of Rel homologues from four different pathogenic bacteria, in order to compare their attributes with well characterized Rel homologues. Here, we present cloning, over-expression, purification and preliminary characterization of these four homologues; and suggest similarities and differences that can be exploited for inhibitor design.


Assuntos
Guanosina Pentafosfato/química , Ligases/química , Pirofosfatases/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Guanosina Pentafosfato/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/patogenicidade , Ligases/genética , Ligases/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Modelos Moleculares , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Pirofosfatases/genética , Pirofosfatases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica
10.
Int J Food Microbiol ; 336: 108912, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-33091754

RESUMO

Listeria monocytogenes contamination in raw pork and ready to eat foods is an important food safety concern, also for the increasing detection of antimicrobial-resistant isolates. Data on L. monocytogenes occurrence, persistence, distribution and genetic characterization in two different plants, namely in continuum from slaughtered pigs, environment and unfinished products (fresh hams) were observed by one-year monitoring and were integrated with their antimicrobial resistance patterns. A total of 98 samples out of the overall 1131 (8.7%) were positive for L. monocytogenes, respectively 2.6% and 13.2% in plants A and B: only three serotypes were identified, 1/2c (50%), 1/2b (36.7%) and 1/2a (13.27%), and strains were classified in 35 pulsotypes and 16 clusters by PFGE; a unique P-type was highlighted according to the detection of virulence genes. The contamination flow of L. monocytogenes has a low occurrence in slaughterhouse (Plant A = 1.1%, Plant B: 3.1%; p > 0.05) and increased throughout the processing chain with trimming area as the most contaminated (Plant A: 25%, Plant B: 57%; (p < 0.05)), both in the environment and in unfinished products (80% in hams before trimming in plant B). The dominant role of environmental contamination in post-slaughter processing is confirmed to be a significant cause of meat contamination by L. monocytogenes. Very high levels of resistance were observed for clindamycin (57%) and high resistance levels (>20-50%) to ciprofloxacin, oxacillin, levofloxacin and daptomycin, confirming the L. monocytogenes resistance trend to a wide range of antimicrobial agents. A total of 11 L. monocytogenes isolates were multidrug resistant and 7 out of them were isolated from slaughtered pigs. An interesting significant (p < 0.05) statistical correlation has been found between resistance to some antimicrobial agents and lineage/serotypes. Microbiological sampling of food and environments after sanitization are commonly used as verification procedure for the absence of L. monocytogenes in food plants and to give assurance of food safety, but strains characterization is necessary for industries to target specific control measures, like the enforcement of the hygiene program and of the control of operator activities, at least for permanent strains. The only presence of L. monocytogenes could not be considered as the conclusive assessment of a potential risk for public health, also in terms of emerging and emerged antimicrobial resistances.


Assuntos
Antibacterianos/farmacologia , Microbiologia de Alimentos , Listeria monocytogenes , Carne de Porco/microbiologia , Matadouros , Animais , Farmacorresistência Bacteriana , Inocuidade dos Alimentos , Genótipo , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Sorogrupo , Suínos , Virulência/genética
11.
Int J Mol Sci ; 21(24)2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33321935

RESUMO

Listeria monocytogenes, an important foodborne pathogen, may be present in different kinds of food and in food processing environments where it can persist for a long time. In this study, 28 L. monocytogenes isolates from fish and fish manufactures were characterized by whole genome sequencing (WGS). Core genome multilocus sequence typing (cgMLST) analysis was applied to compare the present isolates with publicly available genomes of L. monocytogenes strains recovered worldwide from food and from humans with listeriosis. All but one (96.4%) of the examined isolates belonged to molecular serogroup IIa, and one isolate (3.6%) was classified to serogroup IVb. The isolates of group IIa were mainly of MLST sequence types ST121 (13 strains) and ST8 (four strains) whereas the isolate of serogroup IVb was classified to ST1. Strains of serogroup IIa were further subtyped into eight different sublineages with the most numerous being SL121 (13; 48.1% strains) which belonged to six cgMLST types. The majority of strains, irrespective of the genotypic subtype, had the same antimicrobial resistance profile. The cluster analysis identified several molecular clones typical for L. monocytogenes isolated from similar sources in other countries; however, novel molecular cgMLST types not present in the Listeria database were also identified.


Assuntos
Genoma Bacteriano , Listeria monocytogenes/genética , Salmo salar/microbiologia , Truta/microbiologia , Animais , Produtos Pesqueiros/microbiologia , Listeria monocytogenes/classificação , Listeria monocytogenes/patogenicidade , Filogenia , Polônia
12.
Proc Natl Acad Sci U S A ; 117(38): 23774-23781, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32878997

RESUMO

Intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality, and each has evolved specialized strategies to establish and maintain their replicative niche. Listeria monocytogenes is a facultative intracellular pathogen that secretes a pore-forming cytolysin called listeriolysin O (LLO), which disrupts the phagosomal membrane and, thereby, allows the bacteria access to their replicative niche in the cytosol. Nonsynonymous and synonymous mutations in a PEST-like domain near the LLO N terminus cause enhanced LLO translation during intracellular growth, leading to host cell death and loss of virulence. Here, we explore the mechanism of translational control and show that there is extensive codon restriction within the PEST-encoding region of the LLO messenger RNA (mRNA) (hly). This region has considerable complementarity with the 5' UTR and is predicted to form an extensive secondary structure that overlaps the ribosome binding site. Analysis of both 5' UTR and synonymous mutations in the PEST-like domain that are predicted to disrupt the secondary structure resulted in up to a 10,000-fold drop in virulence during mouse infection, while compensatory double mutants restored virulence to WT levels. We showed by dynamic protein radiolabeling that LLO synthesis was growth phase-dependent. These data provide a mechanism to explain how the bacteria regulate translation of LLO to promote translation during starvation in a phagosome while repressing it during growth in the cytosol. These studies also provide a molecular explanation for codon bias at the 5' end of this essential determinant of pathogenesis.


Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Listeria monocytogenes , RNA Bacteriano/química , RNA Mensageiro/química , Regiões 5' não Traduzidas/genética , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Replicação do DNA/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose , Camundongos , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genética
13.
BMC Infect Dis ; 20(1): 559, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32736610

RESUMO

BACKGROUND: Listeriosis is a severe food-borne infection caused by the Gram-positive rod, Listeria monocytogenes. Despite the low incidence (3-8 cases per million), Listeriosis has a case fatality rate of 20-30% as it occurs predominantly in immunocompromised individuals at extremes of age, diabetics and pregnant women. Listeriosis classically presents as a febrile gastroenteritis, isolated bacteremia, meningitis, or maternal-fetal infections. Focal bone and joint infection are rare and primarily involve orthopedic implant devices. Here, we present the first case of Listeria-associated spondylodiscitis. CASE PRESENTATION: A 79-year-old male presents with acute-on-chronic back pain in the absence of risk factors or exposures, aside from age. On radiological imaging, spondylodiscitis of L3-L4 was diagnosed. Subsequently, a CT-guided biopsy was performed to aid in confirming microbiological aetiology. Listeria monocytogenes was grown in culture and patient received appropriate antibacterial therapy. CONCLUSION: The case highlights the utility of image-guided tissue sampling in aiding diagnosis and management in patients with vertebral osteomyelitis. It also encourages consideration of uncommon organisms such as Listeria as an etiology of vertebral osteomyelitis, even in the absence of prosthetic implants.


Assuntos
Discite/diagnóstico por imagem , Listeriose/diagnóstico , Idoso , Antibacterianos/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Dor nas Costas , Bacteriemia/microbiologia , Discite/tratamento farmacológico , Humanos , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Listeriose/tratamento farmacológico , Masculino , Osteomielite/diagnóstico por imagem , Fatores de Risco
14.
PLoS One ; 15(7): e0233945, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701964

RESUMO

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.


Assuntos
Brevibacterium/metabolismo , Queijo/microbiologia , Etanolamina/metabolismo , Microbiologia de Alimentos , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Propilenoglicol/metabolismo , Psychrobacter/metabolismo , Transcriptoma , Aclimatação , Ágar , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Técnicas de Cocultura , Meios de Cultura , Transporte de Elétrons/genética , Fermentação/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Plasmídeos , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Virulência/genética
15.
Int J Food Microbiol ; 333: 108776, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-32693315

RESUMO

We developed an agent-based gastric simulator for a human host to illustrate the within host survival mechanisms of Listeria monocytogenes. The simulator incorporates the gastric physiology and digestion processes that are critical for pathogen survival in the stomach. Mathematical formulations for the pH dynamics, stomach emptying time, and survival probability in the presence of gastric acid are integrated in the simulator to evaluate the portion of ingested bacteria that survives in the stomach and reaches the small intestine. The parameters are estimated using in vitro data relevant to the human stomach and L. monocytogenes. The simulator predicts that 5%-29% of ingested bacteria can survive a human stomach and reach the small intestine. In the absence of extensive scientific experiments, which are not feasible on the grounds of ethical and safety concerns, this simulator may provide a supplementary tool to evaluate pathogen survival and subsequent infection, especially with regards to the ingestion of small doses.


Assuntos
Intestino Delgado/microbiologia , Listeria monocytogenes/patogenicidade , Estômago/microbiologia , Digestão/fisiologia , Ingestão de Alimentos , Ácido Gástrico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos
16.
Appl Environ Microbiol ; 86(17)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32591377

RESUMO

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua, Bacillus subtilis, Lactobacillus plantarum, or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes (inlA, inlB, inlC, inlJ, sigB, prfA, hly, plcA, and plcB) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis, while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response.IMPORTANCE Listeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments.


Assuntos
Fenômenos Fisiológicos Bacterianos , Regulação Bacteriana da Expressão Gênica , Aptidão Genética , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/crescimento & desenvolvimento , Especificidade da Espécie , Transcrição Genética , Virulência/genética
17.
PLoS One ; 15(6): e0234082, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479533

RESUMO

OBJECTIVES: To investigate whether cerebrospinal fluid levels of neuron-specific enolase (CSF-NSE) during the first 72 hours correlate with other tools used to assess ongoing brain damage, including clinical grading of hypoxic-ischemic encephalopathy (HIE), abnormal patterns in amplitude integrated electroencephalography (aEEG), and magnetic resonance imaging (MRI), as well as with the neurodevelopmental outcomes at two years of age. MATERIAL AND METHODS: Prospective observational study performed in two hospitals between 2009 and 2011. Forty-three infants diagnosed with HIE within 6 hours of life were included. HIE was severe in 20 infants, moderate in 12, and mild in 11. Infants with moderate-to-severe HIE received whole-body cooling. Both the HIE cohort and a control group of 59 infants with suspected infection underwent measurement of CSF-NSE concentrations at between 12 and 72 hours after birth. aEEG monitoring was started at admission and brain MRI was performed within the first 2 weeks. Neurodevelopment was assessed at 24 months. RESULTS: The HIE group showed higher levels of CSF-NSE than the control group: median 70 ng/ml (29; 205) vs 10.6 ng/ml (7.7; 12.9); p <0.001. Median levels of CSF-NSE in infants with severe, moderate, and mild HIE were 220.5 ng/ml (120.5; 368.8), 45.5 ng/ml (26, 75.3), and 26 ng/ml (18, 33), respectively. CSF-NSE levels correlated were significantly higher in infants with seizures, abnormal aEEG, or abnormal MRI, compared to those without abnormalities. Infants with an adverse outcome showed higher CSF-NSE levels than those with normal findings (p<0.001), and the most accurate CSF-NSE cutoff level for predicting adverse outcome in the whole cohort was 108 ng/ml and 50ng/ml in surviving infants. CONCLUSIONS: In the era of hypothermia, CSF-NSE concentrations provides valuable information as a clinical surrogate of the severity of hypoxic-ischemic brain damage, and this information may be predictive of abnormal outcome at two years of age.


Assuntos
Lesões Encefálicas/patologia , Hipotermia Induzida/efeitos adversos , Hipóxia-Isquemia Encefálica/diagnóstico , Fosfopiruvato Hidratase/líquido cefalorraquidiano , Lesões Encefálicas/etiologia , Estudos de Casos e Controles , Eletroencefalografia , Feminino , Idade Gestacional , Humanos , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/terapia , Recém-Nascido , Listeria monocytogenes/patogenicidade , Imageamento por Ressonância Magnética , Masculino , Estudos Prospectivos , Convulsões/complicações , Convulsões/diagnóstico , Índice de Gravidade de Doença
19.
PLoS One ; 15(4): e0231393, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352974

RESUMO

Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010-2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon's diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events.


Assuntos
Microbiologia de Alimentos , Variação Genética , Listeria monocytogenes/genética , Virulência/genética , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Humanos , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Listeriose/patologia , Listeriose/transmissão , Tipagem de Sequências Multilocus , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Sorogrupo , Sequenciamento Completo do Genoma
20.
PLoS One ; 15(5): e0232485, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357157

RESUMO

Various produce including cantaloupe, caramel-coated apples, and packaged salads, have been recognized in recent years as vehicles for listeriosis, a human foodborne disease caused by intracellular pathogen Listeria monocytogenes. Our knowledge regarding the role of these foods in L. monocytogenes virulence, however, is limited. Understanding their role in modulating L. monocytogenes virulence can be useful in risk assessments and for developing control measures. In this study, we employed the Galleria mellonella larvae model to evaluate virulence potential of fifteen clinical, environmental and food isolates of L. monocytogenes, related to three major outbreaks, after growth on different foods. The non-human pathogen Listeria innocua was also included in the panel. Strains were inoculated in parallel in 5ml of brain heart infusion (BHI) broth, and on the surfaces of cantaloupe and apple fragments (5g each) at about 105 colony forming units (CFU)/ml/fragment. One set of inoculated broth and food fragments was incubated at 10°C for 5 days while the second set was kept at 25°C for 3 days. L. monocytogenes cells were recovered from the fruits and BHI, washed twice, re-suspended in saline, and used to inoculate G. mellonella larvae at final concentrations of 106 and 105 CFU/larva. The larvae were incubated at 37°C and monitored for mortality (LT50-time taken to kill 50% of the larvae) and phenotypic changes over seven days. L. monocytogenes grown on cantaloupe and apple flesh surfaces resulted in higher virulence than when grown in BHI. L. monocytogenes infection at 106 CFU/larvae resulted in an average LT50 of ≤ 30, 36 and 47 hours on cantaloupe, apples and BHI, respectively. These results represent a 2.5-4-fold increased mortality compared with an LT50 ≥120 hours in larvae infected with the same doses of L. innocua grown in corresponding matrices. Similar trends were also recorded with doses of about 105 CFU /larvae.


Assuntos
Microbiologia de Alimentos , Listeria monocytogenes/patogenicidade , Animais , Carga Bacteriana , Cucumis melo/microbiologia , Meios de Cultura , Doenças Transmitidas por Alimentos/etiologia , Humanos , Larva/microbiologia , Listeria/crescimento & desenvolvimento , Listeria/patogenicidade , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/etiologia , Malus/microbiologia , Modelos Biológicos , Mariposas/microbiologia , Medição de Risco , Virulência
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