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1.
Food Microbiol ; 87: 103367, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948615

RESUMO

Listeria monocytogenes is an important foodborne pathogen, causative agent of listeriosis. The epidemiology and persistence of this bacterium in meat processing plants may be related to its serotype, so it is of utmost importance to carry out a correct differentiation of L. monocytogenes serotypes. The objective of this study was to develop a unique quadruplex real-time quantitative PCR (qPCR) method able to differentiate the four most predominant and worrying L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) in isolates from meat processing plants and ready-to-eat (RTE) dry-cured meat products. The design of specific primers and probes was based on the lmo0737, lmo0308, ORFC (locus genomically equivalent to gltA-gltB) and ORF2110 genes. A qPCR based on a fragment of the 16S rRNA gene was used to ensure the amplification of Listeria spp. genomic DNA. The standard curves showed efficiency values ranging between 92.3% and 105.8% and, R2 values > 0.98. The specificity of the method was also confirmed by the comparison of the results with those obtained by a previously reported conventional multiplex PCR. In addition, none of the strains which were not ascribed to L. monocytogenes amplified any of the target genes related to the four major serotypes of this pathogenic species. The qPCR, therefore, provides a sensitive, specific and rapid tool for identifying the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c and 4b. This method could be very useful for identifying sources of L. monocytogenes contamination in the meat industry or for epidemiological monitoring of persistent strains throughout the processing of RTE meat products.


Assuntos
Listeria monocytogenes/isolamento & purificação , Carne/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Primers do DNA/genética , DNA Bacteriano/genética , Contaminação de Alimentos/análise , Manipulação de Alimentos/instrumentação , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Produtos da Carne/microbiologia , RNA Ribossômico 16S/genética , Sorogrupo
2.
BMC Infect Dis ; 20(1): 83, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996135

RESUMO

BACKGROUND: The more quickly bacterial pathogens responsible for foodborne illness outbreaks can be linked to a vehicle of transmission or a source, the more illnesses can be prevented. Whole genome sequencing (WGS) based approaches to source tracking have greatly increased the speed and resolution with which public health response can pinpoint the vehicle and source of outbreaks. Traditionally, WGS approaches have focused on the culture of an individual isolate before proceeding to DNA extraction and sequencing. For Listeria monocytogenes (Lm), generation of an individual isolate for sequencing typically takes about 6 days. Here we demonstrate that a hybrid, "quasimetagenomic" approach ie; direct sequencing of microbiological enrichments (first step in pathogen detection and recovery) can provide high resolution source tracking sequence data, 5 days earlier than response that focuses on culture and sequencing of an individual isolate. This expedited approach could save lives, prevent illnesses and potentially minimize unnecessary destruction of food. METHODS: Naturally contaminated ice cream (from a 2015 outbreak) was enriched to recover Listeria monocytogenes following protocols outlined in the Bacteriological Analytic Manual (BAM). DNA from enriching microbiota was extracted and sequenced at incremental time-points during the first 48 h of pre-enrichment using the Illumina MiSeq platform (2 by 250), to evaluate genomic coverage of target pathogen, Listeria monocytogenes. RESULTS: Quasimetagenomic sequence data acquired from hour 20 were sufficient to discern whether or not Lm strain/s were part of the ongoing outbreak or not. Genomic data from hours 24, 28, 32, 36, 40, 44, and 48 of pre-enrichments all provided identical phylogenetic source tracking utility to the WGS of individual isolates (which require an additional 5 days to culture). CONCLUSIONS: The speed of this approach (more than twice as fast as current methods) has the potential to reduce the number of illnesses associated with any given outbreak by as many as 75% percent of total cases and potentially with continued optimization of the entire chain of response, contribute to minimized food waste.


Assuntos
Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Sorvetes/microbiologia , Listeria monocytogenes/genética , Listeriose/microbiologia , Metagenômica , Surtos de Doenças , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Listeria monocytogenes/classificação , Listeriose/epidemiologia , Filogenia , Fatores de Tempo , Sequenciamento Completo do Genoma
3.
Int J Food Microbiol ; 314: 108360, 2020 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-31678600

RESUMO

Due to a higher probability for violation of hygiene measures, reconstruction work is a substantial food safety challenge for food business operators (FBOs). Here, we monitored a Listeria monocytogenes contamination scenario during a timely enduring reconstruction period that aimed at an expansion of the main building of a leading meat processing facility. Reconstruction took place while food production was ongoing. We used a longitudinal sampling scheme targeting 40 floor water drains distributed over the food processing environment (FPE) over a five year period. The population structure of L. monocytogenes was determined by PCR-serogrouping, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). While the first sampling deciphered a baseline of contamination (45%), intensified sanitation measures decreased L. monocytogenes prevalence before commencement of work (5%). The reconstruction activities increased the prevalence of L. monocytogenes in the FPE (20.5%) and changed the population structure to a higher proportion of disease-associated genotypes (61%). During the first sampling ST121 was prevalent throughout the FPE, even in the packaging area. After the second and third sampling, following increased application of hypochlorite during sanitation, ST121 was only present in the raw material preparation area. A resilient flora was detected during three sampling events (ST8, ST9 and ST37) which might have not been exposed to daily cleaning in the floor drains. After the accomplishment of reconstruction work, the L. monocytogenes population structure shifted to the condition initially found (45% and 20.5% during the first and sixth sampling event). This paper indicates that reconstruction phases are high risk episodes for food safety in FPEs. Special precautions must be taken to avoid cross-contamination of products since reconstruction is usually ongoing for extended periods of time.


Assuntos
Monitoramento Ambiental , Manipulação de Alimentos , Microbiologia de Alimentos/estatística & dados numéricos , Listeria monocytogenes/isolamento & purificação , Carne/microbiologia , Animais , Arquitetura de Instituições de Saúde , Contaminação de Alimentos/prevenção & controle , Inocuidade dos Alimentos , Genótipo , Listeria monocytogenes/classificação , Listeria monocytogenes/genética
4.
Pol J Microbiol ; 68(3): 353-369, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31880881

RESUMO

Listeria monocytogenes is the etiological factor of listeriosis. The main source of these organisms is food, including dairy products. The aim was to determine the multiple correlations between the drug susceptibility, virulence genes (VGs), and biofilm formation on silicone teat cups of milk-borne and human L. monocytogenes strains. The spread of L. monocytogenes via contaminated teat rubbers was assessed. The L. monocytogenes strains recovered from milk (18), human blood (10), and the reference strain ATCC®19111™ were used in the study. Penicillin resistance was the most prevalent resistance in the milk isolates (n=8; 44.4%), whereas among clinical strains erythromycin resistance was predominating - (n=6; 60%). The most frequent VGs among strains isolated from milk were hlyA (100%) and plcB (100%) whereas in strains isolated from blood - hlyA (100%) and prfA (90%). All tested VGs were present in 50% of blood isolates and 11% of milk-borne strains. The strains isolated from milk formed a significantly stronger biofilm. The strains with more numerous virulence genes were resistant to more antibiotics and formed a stronger biofilm. It was shown that contaminated teat cups might contribute to the transmission of L. monocytogenes in the herd. It seems reasonable to monitor the occurrence of L. monocytogenes biofilm in a dairy processing environment.Listeria monocytogenes is the etiological factor of listeriosis. The main source of these organisms is food, including dairy products. The aim was to determine the multiple correlations between the drug susceptibility, virulence genes (VGs), and biofilm formation on silicone teat cups of milk-borne and human L. monocytogenes strains. The spread of L. monocytogenes via contaminated teat rubbers was assessed. The L. monocytogenes strains recovered from milk (18), human blood (10), and the reference strain ATCC®19111™ were used in the study. Penicillin resistance was the most prevalent resistance in the milk isolates (n=8; 44.4%), whereas among clinical strains erythromycin resistance was predominating ­ (n=6; 60%). The most frequent VGs among strains isolated from milk were hlyA (100%) and plcB (100%) whereas in strains isolated from blood ­ hlyA (100%) and prfA (90%). All tested VGs were present in 50% of blood isolates and 11% of milk-borne strains. The strains isolated from milk formed a significantly stronger biofilm. The strains with more numerous virulence genes were resistant to more antibiotics and formed a stronger biofilm. It was shown that contaminated teat cups might contribute to the transmission of L. monocytogenes in the herd. It seems reasonable to monitor the occurrence of L. monocytogenes biofilm in a dairy processing environment.


Assuntos
Sangue/microbiologia , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia , Leite/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Bovinos , Humanos , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Listeriose/transmissão , Filogenia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
5.
Lett Appl Microbiol ; 69(6): 392-398, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31562639

RESUMO

Four cases of listeriosis in a hospital (A) in New Zealand were identified in 2012. Pulsed-field gel electrophoresis (PFGE) used at the time identified four pulsotypes amongst the clinical isolates. Two of the pulsotypes matched to Listeria monocytogenes isolates obtained from ready-to-eat (RTE) meat samples from a RTE producer tested during a nationwide microbiological survey the month prior. The outbreak investigation confirmed that the RTE producer had supplied product to the hospital and additional testing confirmed the presence of L.  monocytogenes in RTE meats from the hospital kitchen. Two further listeriosis cases presented in another hospital (B) with one clinical isolate identified as the same pulsotype as identified for one case in hospital A, but the epidemiology information concluded that the clinical cases from hospital B were not linked to the outbreak. Retrospective whole-genome sequencing confirmed that epidemiologically linked isolates belonging to three different genotypes for clinical cases from hospital A and RTE meats samples from the hospital kitchen differed by 0-1 core-genome locus or single nucleotide polymorphisms (SNP). The use of core-genome multilocus sequence typing and SNP analysis provided a greater degree of discrimination between isolates compared to PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a listeriosis outbreak associated with a hospital in New Zealand and attributed to contaminated ready-to-eat (RTE) meat supplied to the hospital by a single producer. Retrospective whole-genome sequence analysis of outbreak isolates was found to provide a greater degree of discrimination between isolates compared to pulsed-field gel electrophoresis and supported the conclusions made at the time of the outbreak. The multiple genotypes identified from clinical cases and the RTE meats obtained during the outbreak highlight the importance of epidemiological concordance alongside genotyping.


Assuntos
Infecção Hospitalar/microbiologia , Listeria monocytogenes/genética , Listeriose/epidemiologia , Produtos da Carne/microbiologia , Carne/microbiologia , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado , Microbiologia de Alimentos , Genoma Bacteriano/genética , Genótipo , Humanos , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia , Tipagem de Sequências Multilocus , Nova Zelândia/epidemiologia , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos , Sequenciamento Completo do Genoma
6.
J Med Microbiol ; 68(11): 1677-1685, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31524579

RESUMO

Purpose. Listeria monocytogenes is a foodborne pathogen that causes central nervous system (CNS) and maternal-neonatal (MN) infections, bacteremia (BAC), and gastroenteritis in humans and ruminants. Specific clonal complexes (CC) have been associated with severe listeriosis cases, however, less is known about differences among subgroup virulence patterns. This study aimed to assess variation in virulence across different CC and clinical outcomes.Methodology. Galleria mellonella larvae were used to compare virulence phenotypes of 34 L. monocytogenes strains representing isolates from CC1, CC6 (from lineage I), and CC7, CC9, CC14, CC37 and CC204 (from lineage II) classified by clinical outcome: BAC, CNS and MN infection. Larvae survival, LD50, cytotoxicity, health index scores and bacterial concentrations post-infection were evaluated as quantifiable indicators of virulence.Results. Isolates belonging to CC14 and MN-associated infections are hypervirulent in G. mellonella as they led to lower G. mellonella survival rates and health index scores, as well as reduced cytotoxic effects when compared to other CC and clinical outcomes included here. CC14 isolates also showed increased bacterial concentrations at 8 and 24 h post-infection, indicating ability to survive the initial immune response and proliferate within G. mellonella larvae.Conclusion. Subgroups of L. monocytogenes possess different virulence phenotypes that may be associated with niche-specificity. While hypervirulent clones have been identified so far in lineage I, our data demonstrate that hypervirulent clones are not restricted to lineage I, as CC14 belongs to lineage II. Identification of subgroups with a higher ability to cause disease may facilitate surveillance and management of listeriosis.


Assuntos
Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Animais , Humanos , Larva/microbiologia , Lepidópteros/microbiologia , Listeria monocytogenes/classificação , Listeria monocytogenes/fisiologia , Fenótipo , Virulência
7.
J Dairy Sci ; 102(11): 9674-9688, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31477293

RESUMO

Listeria monocytogenes can survive and grow in a variety of environments, including refrigeration, making it difficult to control and highlighting the importance of optimizing control strategies against this pathogen. Listeria phages are attractive biocontrol agents because phages bind to specific wall teichoic acids (WTA) on the bacterial cell wall, inhibiting pathogens without disrupting the normal microbiota or structure of the food. Common stresses found on dairy products can affect cell wall composition and structure and subsequently affect the efficiency of control strategies that target the cell wall. The goal of this study was to determine the effect of a range of pH and temperatures on the effectiveness of a commercial phage cocktail treatment against several strains of L. monocytogenes in a cheese matrix. We developed a laboratory-scale cheese model that was made at different pH, treated with phage, and then inoculated with L. monocytogenes. Cheeses were incubated at 6, 14, or 22°C for 14 d, and bacterial counts were determined on d 1, 7, and 14. Our data show that phage treatment has a limited ability to reduce L. monocytogenes counts at each temperature tested; however, it was more effective on specific strains of L. monocytogenes when cheese was stored at higher temperatures. More specifically, the average counts of L. monocytogenes on phage-treated cheese stored at 22°C were significantly lower than those on phage-treated cheese stored at 6 or 14°C. Similarly, phage treatment was significantly more effective at inhibiting L. monocytogenes on cheese made at higher pH (6 and 6.5) compared with counts on cheese made at pH 5.5, where L. monocytogenes did not grow. Furthermore, serotype was found to affect the susceptibility of L. monocytogenes to phage treatment; serotype 1/2 strains showed significantly higher susceptibility to phage treatment than serotype 4b strains. Overall, our results suggest the importance of considering the efficacy of phage under conditions (i.e., temperature and pH) specific to a given food matrix when applying interventions against this important foodborne pathogen.


Assuntos
Bacteriófagos , Queijo/microbiologia , Microbiologia de Alimentos , Listeria monocytogenes/virologia , Animais , Carga Bacteriana , Humanos , Concentração de Íons de Hidrogênio , Análise dos Mínimos Quadrados , Listeria monocytogenes/classificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Sorogrupo , Temperatura Ambiente , Fatores de Tempo
8.
Braz J Microbiol ; 50(4): 1063-1073, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31478167

RESUMO

Listeria monocytogenes is one of the most important foodborne pathogens and is a causal agent of listeriosis in humans and animals. The aim of this study was to determine the prevalence, serogroups, antibiotic susceptibility, virulence factor genes, and genetic relatedness of L. monocytogenes strains isolated from 500 poultry samples in Turkey. The isolation sources of 103 L. monocytogenes strains were retail markets (n = 100) and slaughterhouses (n = 3). L. monocytogenes strains were identified as serogroups 1/2a-3a (75.7%, lineage I), 1/2c-3c (14.56%, lineage I), 1/2b-3b-7 (5.82%, lineage II), 4a-4c (2.91%, lineage III), and 4b-4d-4e (0.97%, lineage III). Most of the L. monocytogenes strains (93.2%) were susceptible to the antibiotics tested. PCR analysis indicated that the majority of the strains (95% to 100%) contained most of the virulence genes (hylA, plcA, plcB, prfA, mpl, actA, dltA, fri, flaA inlA, inlC, and inlJ). Pulsed-field gel electrophoresis (PFGE) demonstrated that there were 18 pulsotypes grouped at a similarity of > 90% among the strains. These results indicate that it is necessary to prevent the presence of L. monocytogenes in the poultry-processing environments to help prevent outbreaks of listeriosis and protect public health.


Assuntos
Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeriose/veterinária , Doenças das Aves Domésticas/microbiologia , Matadouros/economia , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Galinhas , Microbiologia de Alimentos , Listeria monocytogenes/classificação , Listeria monocytogenes/efeitos dos fármacos , Listeriose/epidemiologia , Listeriose/microbiologia , Doenças das Aves Domésticas/economia , Doenças das Aves Domésticas/epidemiologia , Prevalência , Turquia/epidemiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
9.
Int J Food Microbiol ; 310: 108289, 2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31487606

RESUMO

This study aimed to characterize serovar 1/2a, 1/2b, 1/2c and 4b of Listeria monocytogenes cultures based on High Resolution Melting (HRM) profiles, targeting 53 fragments in the region comprising prs, Listeria Pathogenicity Island-1 (LIPI-1) and ldh loci, and 28 fragments of inlAB operon. Fifty L. monocytogenes cultures (28 of lineage I, 22 of lineage II) were tested. Real time PCR and EvaGreen-based HRM assays were performed, and the HRM profile for each amplicon was compared to that of L. monocytogenes EGD-e strain. Considering all fragments tested, 45 HRM profiles were identified (Diversity Index = 99.35). Similarity analysis identified two main clusters: the first consisted of 25 cultures, including all 1/2a and 1/2c strains, except for three isolates from food of serovar 4b; the second group only included 1/2b and 4b isolates. Eighteen out of targeted amplicons showed constant HRM characteristics irrespective of the serovar/lineage, and hlyA displayed the most stable melting behavior. Conversely, thirteen amplicons were specific for 1/2b and 4b isolates, showing major variations within actA, followed by prs, prfA, mpl and plcB genes. A fragment targeting an intragenic region (part of plcA and part of an unknown gene) had a melting profile allowing differentiation between 4b and 1/2b isolates showing different Tm variants. Amplicons of inlA and inlB exhibited the largest intra-strain melting differences, and one inlB fragment could allow discriminating between 4b and 1/2b cultures, as well as between lineages. A greater level of genetic diversity amongst 1/2a cultures compared to 1/2c, 1/2b and 4b isolates was observed, with variations identified in LIPI-1, as well as within inlA and inlB genes. Sequencing analysis of amplicons with differential HRM profile from EGD-e confirmed point mutations. The study findings underlines that HRM-based approach may be useful for bacterial subtyping for epidemiological purposes when sequencing-based methods are not available.


Assuntos
Métodos Epidemiológicos , Microbiologia de Alimentos/métodos , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Sorotipagem , Variação Genética , Ilhas Genômicas/genética , Humanos , Listeriose/microbiologia , Filogenia , Reprodutibilidade dos Testes , Sorogrupo
10.
Emerg Microbes Infect ; 8(1): 1195-1204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393224

RESUMO

Listeria monocytogenes is a high risk pathogen which can cause invasive diseases in humans. We previously reported that black-headed gulls from Dianchi Lake of Kunming carrying L. monocytogenes, while the characteristics of these isolates and the relationship with habitats of migratory gulls have not been explored. In this study, we investigated the prevalence and molecular characteristics of Listeria monocytogenes from black-headed gulls in Dianchi Lake, and phylogenetic analysis based on core genome SNPs was used to determine the genetic relationship of the strains from Dianchi Lake and other regions. Occurrence of L. monocytogenes in black-headed gull feces in 2016, 2017 and 2018 was 1.0%, 1.0% and 0.6% respectively. The predominant serotype of 28 isolates was 4b, while the predominant sequence types were ST145 and ST201. Based on their prevalence and genomic relationships, ST5 and ST87 were likely to be sourced locally while ST145 and ST201 were likely to be non-local. L. monocytogenes may travel along the bird migration route leading to transmission over a large geographical span carried by black-headed gull. Although the prevalence of L. monocytogenes was low, its carriage by the migratory black-headed gulls poses potential public health risks in regions where the migratory birds passage and reside.


Assuntos
Doenças das Aves/microbiologia , Portador Sadio/veterinária , Charadriiformes , Transmissão de Doença Infecciosa , Leptospirose/veterinária , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Animais , Portador Sadio/microbiologia , China , Variação Genética , Genótipo , Lagos , Leptospirose/microbiologia , Listeria monocytogenes/genética , Epidemiologia Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Prevalência , Análise de Sequência de DNA , Sorogrupo
11.
J Appl Microbiol ; 127(5): 1349-1361, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31432571

RESUMO

AIMS: An extensive source investigation was conducted on a dairy farm with neurolisteriosis and subclinical mastitis cases to identify infection source and potential transmission routes of Listeria monocytogenes. METHODS AND RESULTS: A total of 36 L. monocytogenes isolates were obtained from animal clinical cases (neurolisteriosis and udder infection) and the farm environment (silage, faeces, water). Isolates were typed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Their virulence potential was assessed using the gentamicin protection assay and WGS-based identification of virulence genes. PFGE and WGS revealed a high genetic diversity of L. monocytogenes. An epidemiological link was confirmed for isolates from (i) several subclinical mastitis cases, (ii) silage and subclinical mastitis cases and (iii) different water sources. The neurolisteriosis isolate belonged to clonal complex (CC) 1, but infection source was not identified. A high occurrence (9/47 cows; 19·1%) of subclinical mastitis was observed with isolates belonging to CC2, CC4 and CC11. CONCLUSIONS: The dairy farm environment was contaminated with diverse L. monocytogenes strains, including genotypes associated with human disease. Several isolates harboured genetic determinants associated with increased infectious potential in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that subclinical listerial mastitis should not be neglected as a potential source of milk contamination. The presence of hypervirulent CCs in subclinical mastitis cases calls for the implementation of improved mastitis detection.


Assuntos
Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Mastite Bovina/epidemiologia , Mastite Bovina/microbiologia , Meningite por Listeria/veterinária , Animais , Bovinos , Fazendas , Fezes/microbiologia , Feminino , Genótipo , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Meningite por Listeria/epidemiologia , Meningite por Listeria/microbiologia , Silagem/microbiologia , Virulência/genética
12.
Int J Mol Sci ; 20(17)2019 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-31450632

RESUMO

The pathogenic Gram-positive bacterium Listeria monocytogenes has been evolving into a few phylogenetic lineages. Phylogenetically defined substitutions were described in the L. monocytogenes virulence factor InlB, which mediates active invasion into mammalian cells via interactions with surface receptors c-Met and gC1q-R. InlB internalin domain (idInlB) is central to interactions with c-Met. Here we compared activity of purified recombinant idInlB isoforms characteristic for L. monocytogenes phylogenetic lineage I and II. Size exclusion chromatography and intrinsic fluorescence were used to characterize idInlBs. Western blotting was used to study activation of c-Met-dependent MAPK- and PI3K/Akt-pathways. Solid-phase microplate binding and competition assay was used to quantify interactions with gCq1-R. Isogenic recombinant L. monocytogenes strains were used to elucidate the input of idInlB isoforms in HEp-2 cell invasion. Physicochemical parameters of idInlB isoforms were similar but not identical. Kinetics of Erk1/2 and Akt phosphorylation in response to purified idInlBs was lineage specific. Lineage I but not lineage II idInlB specifically bound gC1q-R. Antibody against gC1q-R amino acids 221-249 inhibited invasion of L. monocytogenes carrying lineage I but not lineage II idInlB. Taken together, obtained results suggested that phylogenetically defined substitutions in idInlB provide functional distinctions and might be involved in phylogenetically determined differences in virulence potential.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Listeria monocytogenes/classificação , Listeria monocytogenes/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Filogenia , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Bactérias/química , Linhagem Celular , Humanos , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Fatores de Virulência
13.
J Zoo Wildl Med ; 50(1): 183-189, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31120677

RESUMO

Listeria monocytogenes is a ubiquitous environmental bacterium that causes disease in a wide range of species. Infection with this pathogen is most frequently diagnosed in ruminant livestock, but is also known to infect people and occasionally wildlife. Postmortem examinations of Western European hedgehogs (Erinaceus europaeus) in Great Britain (2011-2017) identified five (5/266, 2%, 95% confidence interval: 0.8-4.3%) animals with L. monocytogenes infection. The L. monocytogenes isolates comprised three serogroup 1/2a and two serogroup 4 from three multilocus sequence types (2, 37, and 121), all of which were different by single-nucleotide polymorphism analysis, indicating they were distinct and epidemiologically unrelated. These findings are consistent with hedgehogs contracting sporadic infection from the environment, perhaps through eating soil-dwelling invertebrates. Examination of data from scanning surveillance programs focused on other British wildlife species indicates that the hedgehog is one of the wildlife species from which L. monocytogenes has been most frequently identified to date in Great Britain. However, further studies of multiple taxa with comparable sampling efforts are required to assess the relative frequency of L. monocytogenes infection in different wildlife species. The bacterium was isolated from extraintestinal sites in multiple hedgehogs, which may indicate septicemia. However, histological examination was limited and could not discriminate subclinical infection from disease (i.e., listeriosis). Although L. monocytogenes is a zoonotic pathogen, disease in people is typically contracted from the ingestion of contaminated foods. The risk to immunocompetent people of contracting listeriosis from hedgehogs is considered very low to negligible.


Assuntos
Ouriços-Cacheiros , Listeria monocytogenes/fisiologia , Listeriose/veterinária , Animais , Autopsia/veterinária , Feminino , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia , Listeriose/patologia , Masculino , Reino Unido
14.
Immunopharmacol Immunotoxicol ; 41(2): 292-298, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31046503

RESUMO

Objective: The current therapeutic regimens for tuberculosis (TB) are complex and involve the prolonged use of multiple antibiotics with diverse side effects that lead to therapeutic failure and bacterial resistance. The standard appliance of immunotherapy may aid as a powerful tool to combat the ensuing threat of TB. We have earlier reported the immunotherapeutic potential of N-formylated peptides of two secretory proteins of Mycobacterium tuberculosis H37Rv. Here, we investigated the immunotherapeutic effect of an N-formylated peptide from Listeria monocytogenes in experimental TB. Methods: The N-terminally formylated listerial peptide with amino acid sequence 'f-MIGWII' was tested for its adjunctive therapeutic efficacy in combination with anti-tuberculosis drugs (ATDs) in the mouse model of TB. In addition, its potential to generate reactive oxygen species (ROS) in murine neutrophils was also evaluated. Results: The LemA peptide (f-MIGWII) induced a significant increase in the intracellular ROS levels of mouse neutrophils (p ≤ .05). The ATD treatment reduced the colony forming units (CFU) in lungs and spleen of infected mice by 2.39 and 1.67 log10 units, respectively (p < .001). Treatment of the infected mice with combination of ATDs and LemA peptide elicited higher therapeutic efficacy over ATDs alone. The histopathological changes in the lungs of infected mice also correlated well with the CFU data. Conclusions: Our results clearly indicate that LemA peptide conferred an additional therapeutic effect when given in combination with the ATDss (p < .01) and hence can be used as adjunct to the conventional chemotherapy against TB.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/farmacologia , Listeria monocytogenes/classificação , Oligopeptídeos/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Animais , Proteínas de Bactérias/química , Quimioterapia Combinada , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Neutrófilos/patologia , Oligopeptídeos/química , Espécies Reativas de Oxigênio/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia
15.
Braz J Microbiol ; 50(3): 817-824, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30976991

RESUMO

This study focuses on the prevalence of Listeria monocytogenes (Lm) in pork meat and on inert surfaces from slaughterhouses in Sonora, Mexico. A total of 21 Lm were obtained from 103 samples, giving a prevalence of 20.3%. The prevalence of Lm in pork loin was 15.9% and 20.8% for inert surfaces in Federal Inspection Type (FIT) slaughterhouses. For non-FIT slaughterhouses, the prevalence was 25.7%. PCR amplification of genomic DNA from the Lm isolates revealed the presence of the hlyA gene, suggesting a pathogenic nature for these isolates. The isolates obtained in this work all clustered with Lm, according to our phylogenetic analysis based on the 16S rDNA sequence. This Lm cluster indicates that Lm isolates 7-2, 4, 2-1, 10B, 8, 3, 3-3, and 9 share 16S rRNA identity with other Lm isolates that have been reported as foodborne pathogens (rR2-502, J1817, J1816, J1926) and that are involved in foodborne outbreaks. The most commonly detected serotypes were 1/2a and 1/2b. All isolates displayed differential responses to the assayed antibiotics, and most isolates were able to grow in the presence of penicillin G, or both penicillin and penicillin-derived (oxacillin) antibiotics.


Assuntos
Manipulação de Alimentos/instrumentação , Listeria monocytogenes/isolamento & purificação , Carne Vermelha/microbiologia , Matadouros/estatística & dados numéricos , Animais , Antibacterianos/farmacologia , Contaminação de Alimentos/análise , Listeria monocytogenes/classificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , México , Testes de Sensibilidade Microbiana , Filogenia , Suínos
16.
Int J Food Microbiol ; 299: 23-32, 2019 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-30939364

RESUMO

Listeria monocytogenes is sporadically detected on a range of ready to eat fresh produce lines, such as spinach and rocket, and is a threat to public health. However, little is known about the diversity of L. monocytogenes present on fresh produce and their potential pathogenicity. In this work, fifteen Listeria monocytogenes isolates from the UK fresh produce supply chain were characterised using whole genome sequencing (WGS). Additionally, isolates were characterised based on their ability to form biofilm. Whole genome sequencing data was used to determine the sequence type of isolates based on multi-locus sequence typing (MLST), construct a core single nucleotide polymorphism (SNP) phylogeny and determine the presence of virulence and resistance associated genes. MLST revealed 9 distinct sequence types (STs) spanning 2 lineages (I & II) with one isolate belonging to the ST6 subtype, strains from which have been recently implicated in two large, food-associated L. monocytogenes outbreaks in South Africa and across Europe. Although most of the 15 isolates were different, comparison of core genome SNPs showed 4 pairs of 'indistinguishable' strains (<5 SNPs difference). Virulence profiling revealed that some isolates completely lacked the Listeria pathogenicity island-3 (LIPI-3) amongst other virulence factors. Investigation of the inlA gene showed that no strains in this study contained a premature stop codon (PMSC), an indicator of attenuated virulence. Assessment of biofilm production showed that isolates found in the fresh produce supply chain differ in their ability to form biofilm. This trait is considered important for L. monocytogenes to persist in environments associated with food production and processing. Overall the work indicates that a genetically diverse range of L. monocytogenes strains is present in the UK fresh produce supply chain and the virulence profiles found suggests that at least some of the strains are capable of causing human illness. Interestingly, the presence of some genetically indistinguishable isolates within the 15 isolates examined suggests that cross-contamination in the fresh produce environment does occur. These findings have useful implications in terms of food safety and for informing microbial surveillance programmes in the UK fresh produce supply chain.


Assuntos
Microbiologia de Alimentos , Inocuidade dos Alimentos , Listeria monocytogenes/classificação , Verduras/microbiologia , Proteínas de Bactérias/genética , Códon sem Sentido , Farmacorresistência Bacteriana/genética , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Listeriose/transmissão , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Reino Unido , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
17.
Emerg Microbes Infect ; 8(1): 17-28, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866756

RESUMO

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.


Assuntos
Quitinases/genética , Surtos de Doenças , Gastroenterite/microbiologia , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeriose/epidemiologia , Adolescente , Adulto , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Células CACO-2 , Criança , Pré-Escolar , Códon de Terminação , Feminino , Microbiologia de Alimentos , Gastroenterite/epidemiologia , Genômica , Alemanha/epidemiologia , Células HeLa , Células Hep G2 , Humanos , Lactente , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Filogenia , Fatores de Virulência/genética , Adulto Jovem
18.
J Appl Microbiol ; 126(5): 1373-1382, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30835952

RESUMO

AIMS: The aim of this study was to evaluate the genetic diversity and resistance phenotypes of Listeria monocytogenes strains isolated from clinical encephalitis cases, and compare this population to isolates derived from tank milk of healthy animals. METHODS AND RESULTS: A total of 57 L. monocytogenes strains isolated from ruminant's listeriosis cases (n = 31) and from tank milk of healthy ruminants (n = 26) were characterized by species PCR, molecular serotyping, PCR detection of virulence genes, pulsed-field gel electrophoresis and antimicrobial susceptibility testing. All strains possessed inlA, inlC, inlJ, plcA, actA, hlyA and iap virulence-associated genes while serotyping analysis revealed that they were mainly assigned into IVb group. Genotyping revealed 50 pulsotypes among the 57 strains assigned into seven clusters while indistinguishable pulsotypes between clinical and milk strains were not identified. Resistance of L. monocytogenes isolates to 14-16 antimicrobial agents tested was observed and 23 antimicrobial resistance profiles (ARPs) were defined while no apparent predominant ARP type was observed among isolates. CONCLUSIONS: Small ruminants are exposed to a broad range of antimicrobial-resistant as well as genetically diverse strains of L. monocytogenes carrying virulence-associated genes but not all of them associated with the disease. Pulsed-field gel electrophoresis analysis suggests that pulsotypes associated with encephalitis are found in farms only in association with listeriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are valuable in understanding the ecology of this important food-borne pathogen and creating awareness for the emerging antimicrobial resistance.


Assuntos
Encefalite Infecciosa/microbiologia , Listeria monocytogenes/classificação , Listeriose/microbiologia , Ruminantes/microbiologia , Animais , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Grécia , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Leite/microbiologia , Sorotipagem , Fatores de Virulência/genética
20.
Microb Genom ; 5(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30775964

RESUMO

We present the LiSEQ (Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.


Assuntos
Laticínios/microbiologia , Produtos Pesqueiros/microbiologia , Listeria monocytogenes/classificação , Listeriose/microbiologia , Produtos da Carne/microbiologia , Animais , Estudos Transversais , Farmacorresistência Bacteriana/genética , Europa (Continente) , Manipulação de Alimentos , Microbiologia de Alimentos , Variação Genética , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Virulência/genética , Sequenciamento Completo do Genoma
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