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1.
Food Microbiol ; 85: 103280, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31500706

RESUMEN

Listeria monocytogenes causes severe diseases in humans, including febrile gastroenteritis and systemic infections that has a high mortality despite antibiotic treatment. This pathogen may cause massive outbreaks associated to the consumption of contaminated food products, which highlight its importance in public health. In the last decade, L. monocytogenes has emerged as a foodborne pathogen of major importance in Chile. A previous work showed that in Chile during 2008 and 2009, L. monocytogenes serotypes 1/2a, 1/2b and 4b were the most frequently identified in food and clinical strains. Here we report the molecular characterization of L. monocytogenes strains isolated from 2008 to 2017 in the country. Our results indicate that serotypes 1/2a, 1/2b and 4b continue to be the most commonly found in food products. In addition, we identify persistent and widespread PFGE subtypes. This study reports ten years of epidemiological surveillance ofL. monocytogenes in Chile.


Asunto(s)
Monitoreo Epidemiológico , Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos/epidemiología , Listeria monocytogenes/genética , Listeriosis/epidemiología , Chile/epidemiología , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos/microbiología , Gastroenteritis/epidemiología , Gastroenteritis/microbiología , Variación Genética , Humanos , Listeria monocytogenes/patogenicidad , Productos de la Carne/microbiología , Epidemiología Molecular , Salud Pública , Serogrupo , Serotipificación , Factores de Virulencia/genética
2.
Food Microbiol ; 85: 103284, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31500712

RESUMEN

The GAD system is widely present in several types of organisms and is known to play an important role in bacterial acid tolerance. There is only one account of this system playing a role in oxidative stress in bacteria and one in yeasts. Here we show for first time that it affects the oxidative stress resistance of a Gram-positive bacterium, (L. monocytogenes, tested in three strains; 10403S, EGD-e, and LO28). We found a statistically significant reduction in survival after H2O2 exposure in ΔgadD3 and ΔgadD2 of EGD-e and in ΔgadD1 of LO28. Furthermore, we observed a lag phase prolongation in ΔgadD3 of 10403S and EGD-e and a larger inhibition zone in disk diffusion assay for ΔgadD1 and ΔgadD3 of EGD-e upon H2O2 exposure. All GAD genes playing a role in oxidative stress resistance are part of GADi system and this occurs partly through catalase activity, while the most potent GADe system plays no role. The latter effects could occur through the GABA shunt, but we show here that mutants in succinate semialdehyde dehydrogenase do not show a phenotype suggesting that either effects are through the GABA transaminase or, this pathway is not involved. Our study highlights for first time the role of the GAD system in oxidative stress resistance of a Gram-positive bacterium, which could be used in Food Hurdle Technology to eliminate pathogens such as L. monocytogenes, while it gives an insight on the general mechanism.


Asunto(s)
Glutamato Descarboxilasa/metabolismo , Peróxido de Hidrógeno/farmacología , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/enzimología , Estrés Oxidativo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Glutamato Descarboxilasa/genética , Concentración de Iones de Hidrógeno , Listeria monocytogenes/genética
3.
Pol J Microbiol ; 68(3): 353-369, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31880881

RESUMEN

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.


Asunto(s)
Sangre/microbiología , Listeria monocytogenes/aislamiento & purificación , Listeriosis/microbiología , Leche/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Bovinos , Humanos , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Listeria monocytogenes/fisiología , Listeriosis/transmisión , Filogenia , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
4.
Infect Immun ; 88(1)2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31685546

RESUMEN

Listeria monocytogenes, a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here, we demonstrate that DHNA is essential for virulence in a murine model of listeriosis due to both respiration-dependent and -independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five ΔmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent ΔmenD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytoI (lmo1576). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA-deficient L. monocytogenes RNA-seq transcriptome analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI strain than in the wild-type strain, whereas two genes (lmo1917 and lmo2103) demonstrated lower expression in the ΔytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate, and lactate flux were altered in a ΔytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of L. monocytogenes Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes.


Asunto(s)
Listeria monocytogenes/metabolismo , Listeriosis/microbiología , Proteínas Mutantes/metabolismo , Naftoles/metabolismo , Supresión Genética , Factores de Transcripción/metabolismo , Animales , Citosol/química , Modelos Animales de Enfermedad , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/patogenicidad , Ratones , Viabilidad Microbiana , Proteínas Mutantes/genética , Factores de Transcripción/genética , Virulencia , Vitamina K 2/análisis , Secuenciación Completa del Genoma
5.
Nat Commun ; 10(1): 4283, 2019 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-31570766

RESUMEN

The foodborne pathogen Listeria monocytogenes (Lm) is a highly heterogeneous species and currently comprises of 4 evolutionarily distinct lineages. Here, we characterize isolates from severe ovine listeriosis outbreaks that represent a hybrid sub-lineage of the major lineage II (HSL-II) and serotype 4h. HSL-II isolates are highly virulent and exhibit higher organ colonization capacities than well-characterized hypervirulent strains of Lm in an orogastric mouse infection model. The isolates harbour both the Lm Pathogenicity Island (LIPI)-1 and a truncated LIPI-2 locus, encoding sphingomyelinase (SmcL), a virulence factor required for invasion and bacterial translocation from the gut, and other non-contiguous chromosomal segments from another pathogenic species, L. ivanovii. HSL-II isolates exhibit a unique wall teichoic acid (WTA) structure essential for resistance to antimicrobial peptides, bacterial invasion and virulence. The discovery of isolates harbouring pan-species virulence genes of the genus Listeria warrants global efforts to identify further hypervirulent lineages of Lm.


Asunto(s)
Listeria monocytogenes/genética , Listeriosis/microbiología , Animales , Células CACO-2 , Genoma Bacteriano , Genómica , Cabras/microbiología , Humanos , Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/patogenicidad , Ratones , Filogenia , Porcinos/microbiología , Virulencia
6.
BMC Infect Dis ; 19(1): 893, 2019 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-31655547

RESUMEN

BACKGROUND: Neonatal listeriosis is a rare but severe disease manifesting as septicemia and central nervous system (CNS) infections with a high fatality rate of around 20 to 30%. Whole genome sequencing (WGS) is a promising technique for pathogen identification and infection source tracing with its high resolution. CASE PRESENTATION: A case of neonatal sepsis with listeriosis was reported with positive blood culture for Listeria monocytogenes. The case was investigated to confirm the vertical transmission of the infection and identify the potential food source of the maternal L. monocytogenes infection using WGS. L. monocytogenes was isolated from the neonate's blood sample the day after caesarean delivery and from the mother's genital and pudenda swab samples 5 days and 13 days after caesarean delivery. WGS showed that the isolate from the neonate was identical to the genome type of the isolates from the mother, with only one of the 4 isolates from the mother differing by one single nucleotide polymorphism (SNP). By WGS, one L. monocytogenes isolate from a ready-to-eat (RTE) meat sample in the patients' community market shared the same sequence type but was ruled out as the cause of infection, with 57 SNP differences to the strain causing the maternal-neonatal infection. The food isolate also carried a novel plasmid pLM1686 that harbored heavy metal resistance genes. After caesarean section, the mother was treated with a third generation cephalosporin which L. monocytogenes is naturally resistant to, which may explain why genital and pudenda swabs were still culture-positive for L. monocytogenes 13 days after delivery. CONCLUSIONS: Genital swab culture for L. monocytogenes had been informative in the diagnosis of maternal listeriosis in this case. The high resolution of WGS confirmed the maternal-neonatal transmission of L. monocytogenes infection and ruled out the L. monocytogenes contaminated RTE meat from the local market as the direct source of the mother's infection.


Asunto(s)
Listeriosis/diagnóstico , Listeriosis/genética , Sepsis Neonatal/microbiología , China , Femenino , Contaminación de Alimentos , Microbiología de Alimentos , Humanos , Recién Nacido , Enfermedades del Recién Nacido/microbiología , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Listeriosis/transmisión , Carne/microbiología , Sepsis Neonatal/tratamiento farmacológico , Polimorfismo de Nucleótido Simple , Embarazo , Secuenciación Completa del Genoma , Adulto Joven
7.
Lett Appl Microbiol ; 69(6): 392-398, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31562639

RESUMEN

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.


Asunto(s)
Infección Hospitalaria/microbiología , Listeria monocytogenes/genética , Listeriosis/epidemiología , Productos de la Carne/microbiología , Carne/microbiología , Brotes de Enfermedades , Electroforesis en Gel de Campo Pulsado , Microbiología de Alimentos , Genoma Bacteriano/genética , Genotipo , Humanos , Listeria monocytogenes/clasificación , Listeria monocytogenes/aislamiento & purificación , Listeriosis/microbiología , Tipificación de Secuencias Multilocus , Nueva Zelanda/epidemiología , Polimorfismo de Nucleótido Simple/genética , Estudios Retrospectivos , Secuenciación Completa del Genoma
8.
Nat Biotechnol ; 37(12): 1493-1501, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31548729

RESUMEN

Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.


Asunto(s)
Sistemas CRISPR-Cas/genética , Ingeniería Genética/métodos , Transcripción Genética/genética , Escherichia coli/genética , Células HEK293 , Humanos , Listeria monocytogenes/genética , ARN Guia/genética
9.
Curr Microbiol ; 76(12): 1425-1434, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31552450

RESUMEN

In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.


Asunto(s)
Marcadores de Afinidad/química , Azidas/química , Técnicas Bacteriológicas/métodos , ADN Bacteriano/química , Listeria monocytogenes/fisiología , Viabilidad Microbiana , Recuento de Colonia Microbiana , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Microbiología del Suelo
10.
Can J Microbiol ; 65(12): 913-921, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31491332

RESUMEN

This study reports the use of reverse transcription - loop-mediated isothermal amplification (RT-LAMP) to detect Listeria monocytogenes in meat. The assay was designed to target the iap gene of L. monocytogenes, to which four primers, recognizing six distinct iap sites, were designed. We optimized the RT-LAMP conditions and established the following optimal systems: 60 min, 63 °C, 2.0 mmol/L MgSO4, 1.0 mol/L betaine, 2.0 mmol/L dNTPs, 320 U/mL Bst DNA polymerase, 0.4 µmol/L outer primers, and 0.8 µmol/L inner primers. The RT-LAMP amplification products were identified by a visible white Mg2P2O7 precipitate or electrophoresis on a 2% agarose gel. RT-LAMP has a sensitivity of 7.3 × 101 CFU/mL, which is 2-fold higher than that of LAMP. When commercially available raw meat samples (including beef, pork, mutton, and rabbit) were analyzed simultaneously with RT-LAMP and the Chinese National Standard GB 4789.30-2016, their abilities to detect L. monocytogenes were the same. Samples containing L. monocytogenes killed by 15 psi at 121 °C for 15 min were used to confirm the specificity of RT-LAMP for live microorganisms. Thus, we used RT-LAMP to efficiently detect L. monocytogenes in meat products.


Asunto(s)
Microbiología de Alimentos/métodos , Listeria monocytogenes/aislamiento & purificación , Productos de la Carne/microbiología , Técnicas de Amplificación de Ácido Nucleico , Animales , Proteínas Bacterianas/genética , Lipoproteínas/genética , Listeria monocytogenes/genética , Carne/microbiología , Sensibilidad y Especificidad
11.
Int J Food Microbiol ; 310: 108289, 2019 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-31487606

RESUMEN

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.


Asunto(s)
Métodos Epidemiológicos , Microbiología de Alimentos/métodos , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Serotipificación , Variación Genética , Islas Genómicas/genética , Humanos , Listeriosis/microbiología , Filogenia , Reproducibilidad de los Resultados , Serogrupo
12.
J Appl Microbiol ; 127(5): 1349-1361, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31432571

RESUMEN

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.


Asunto(s)
Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/patogenicidad , Mastitis Bovina/epidemiología , Mastitis Bovina/microbiología , Meningitis por Listeria/veterinaria , Animales , Bovinos , Granjas , Heces/microbiología , Femenino , Genotipo , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Meningitis por Listeria/epidemiología , Meningitis por Listeria/microbiología , Ensilaje/microbiología , Virulencia/genética
13.
Food Microbiol ; 84: 103239, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31421769

RESUMEN

Bacteriophage-based biocontrols are one of several tools available to control Listeria monocytogenes in food and food processing environments. The objective of this study was to determine if phage-resistance that has been characterized with a select few Listeria phages would also confer resistance to a diverse collection of over 100 other Listeria phages. We show that some mutations that are likely to emerge in bacteriophage-treated populations of serotype 1/2a L. monocytogenes can lead to cross-resistance against almost all types of characterized Listeria phages. Out of the 120 phages that showed activity against the parental strain, only one could form visible plaques on the mutant strain of L. monocytogenes lacking rhamnose in its wall teichoic acids. An additional two phages showed signs of lytic activity against this mutant strain; although no visible plaques were observed. The findings presented here are consistent with other studies showing mutations conferring phage resistance through loss of rhamnose likely pose the greatest challenge for phage-based biocontrol in serotype 1/2a strains.


Asunto(s)
Bacteriófagos/fisiología , Listeria monocytogenes/genética , Listeria monocytogenes/virología , Mutación , Agentes de Control Biológico , Manipulación de Alimentos/métodos , Inocuidad de los Alimentos/métodos , Serogrupo
14.
Food Microbiol ; 84: 103234, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31421784

RESUMEN

Listeria monocytogenes is a relevant pathogen usually associated with meat and ready-to-eat products. This study aimed to assess the distribution, adhesion, virulence and antibiotic resistance of L. monocytogenes in a pork production chain. Environment, carcass and food samples (n = 894) were obtained from different steps of a pork production chain over a 6-month period (10 samplings), including from farms and the slaughterhouse (reception, slaughtering, processing, storage and end products). L. monocytogenes was detected in samples from the reception (lairage floor, 1/10), slaughtering (drains, 2/20) and cutting room stages (conveyor belts in the final packing stage - 11/20, knife - 1/40, and cutting boards - 1/20). Positive results for conveyor belts were recorded in seven consecutive samplings. L. monocytogenes isolates (n = 87) were characterized as belonging to serogroup IVb and presented positive PCR results for inlA, inlB, inlC, inlJ, hlyA, plcA, actA and iap. Isolates were selected according to the original samples (n = 31) and subjected to Pulsed Field Gel Electrophoresis (PFGE), demonstrating their high clonal identity (98.4-100%). According to PFGE results and their original samples, isolates were selected (n = 16) and subjected to phenotypic assay to assess their adhesion potential and tested for resistance against 15 antibiotics; all tested isolates presented weak adhesion potential and were resistant to ampicillin. The present study demonstrated the persistence of L. monocytogenes in the pork processing facility, indicating the potential risk for cross-contamination with a potential virulent and resistant clone.


Asunto(s)
Mataderos , Adhesión Bacteriana , Microbiología de Alimentos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/patogenicidad , /microbiología , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Brasil , Farmacorresistencia Bacteriana Múltiple , Granjas , Listeria monocytogenes/genética , Porcinos , Virulencia
15.
Emerg Microbes Infect ; 8(1): 1195-1204, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31393224

RESUMEN

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.


Asunto(s)
Enfermedades de las Aves/microbiología , Portador Sano/veterinaria , Charadriiformes , Transmisión de Enfermedad Infecciosa , Leptospirosis/veterinaria , Listeria monocytogenes/clasificación , Listeria monocytogenes/aislamiento & purificación , Animales , Portador Sano/microbiología , China , Variación Genética , Genotipo , Lagos , Leptospirosis/microbiología , Listeria monocytogenes/genética , Epidemiología Molecular , Filogenia , Polimorfismo de Nucleótido Simple , Prevalencia , Análisis de Secuencia de ADN , Serogrupo
16.
Lett Appl Microbiol ; 69(4): 286-293, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31392736

RESUMEN

The prevalence of Listeria monocytogenes in the retail fish markets of the Kerala, India was investigated by screening 227 samples comprising of marine finfish (n = 97) shellfish (n = 19), ready-to-cook fish products (n = 47), ready-to-eat fish products (n = 10), dried fish (n = 11) and retail ice (n = 43). The prevalence of L. monocytogenes and L. innocua was 2·7% and 17·2% respectively. Sample category wise, prevalence of L. monocytogenes was higher in marine finfish (1·8%) and retail ice (0·9%). All the L. monocytogenes isolates carried virulent genes namely inlA, inlC, inlJ, hlyA, iap, plcA, prfA genes and majority (82%) belonged to 1/2a, 3a serogroups. L. monocytogenes isolates were multidrug-resistant and showed resistance to ampicillin, penicillin, erythromycin, tetracycline and clindamycin. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) delineated 58% genetic heterogeneity among the L. monocytogenes strains. The study reports that genetic similarities of the isolates were interlinked to their serogroup and sample origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of Listeria monocytogenes, in the retail fish markets of Kerala, India was low but their relatively higher presence in marine finfish and retail ice and virulent nature of the isolates signifies food safety concerns. Moreover, multidrug-resistant nature of these isolates may potentially lead to spread of antimicrobial resistance. This study identified retail ice as a vehicle for entry of L. monocytogenes in retail fish and hence, there is a need to ensure quality of retail ice used for maintaining the cold-chain.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Listeria monocytogenes/efectos de los fármacos , Alimentos Marinos/microbiología , Mariscos/microbiología , Ampicilina/farmacología , Animales , Clindamicina/farmacología , Eritromicina/farmacología , Explotaciones Pesqueras , Peces/microbiología , Microbiología de Alimentos , Inocuidad de los Alimentos , Heterogeneidad Genética , Hielo/análisis , India , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Penicilinas/farmacología , Prevalencia , Serogrupo , Tetraciclina/farmacología
17.
Zhonghua Er Ke Za Zhi ; 57(8): 603-607, 2019 Aug 02.
Artículo en Chino | MEDLINE | ID: mdl-31352745

RESUMEN

Objective: To summarize the clinical characteristics of Listeria monocytogenes meningitis (LMM) with complications, and analyze the outcomes of next generation sequencing. Methods: Clinical characteristics, laboratory findings, imaging features, antibiotics treatment, and next generation sequencing of cerebrospinal fluid were analyzed in 3 LMM patients who were hospitalized in the Department of Infectious Diseases of Beijing Children's Hospital Affiliated to Capital Medical University from July 2015 to November 2017. Results: The three patients were 1-year-old girl, 2-year-old girl, and 9-year-old boy, with normal immune function. They had eaten refrigerated food, milk or dairy products before onset. Symptoms included fever, headache, abdominal pain, diarrhea, vomiting, and convulsions, etc. The complications of two cases (case 2 and 3) were appendicitis and Meckel's diverticulitis. The other one (case 1) was with sepsis and pneumonia. Leukocyte counts in cerebrospinal fluid were elevated in all the three cases, and cranial magnetic resonance imaging showed meningeal or periventricular involvement. All the children were diagnosed with LMM by positive CSF culture. CSF for next generation sequencing was sent after carbapenem antibiotics using, yet all the results were positive. The positive results were returned 2, 9, and 9 days earlier than culture results, respectively. The gene coverage was 5.00%, 7.00%, 0.04%, and the reads was 2 561, 1 011 and 8, respectively. All the three children had recurrent fever despite using cephalosporin. Levels of leukocytes in the blood and CSF further elevated. After using carbapenem antibiotics, patients improved eventually and were discharged from hospital. Conclusions: LMM can occur in children with normal immune function and is usually associated with digestive system symptoms. Listeria monocytogenes can be detected quickly and accurately by the next generation sequencing technology, without being limited to sampling time and antibiotics application.


Asunto(s)
Listeria monocytogenes/genética , Meningitis por Listeria/líquido cefalorraquídeo , Antibacterianos/uso terapéutico , Niño , Preescolar , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante , Listeria monocytogenes/aislamiento & purificación , Imagen por Resonancia Magnética , Masculino , Meningitis por Listeria/tratamiento farmacológico
18.
Lett Appl Microbiol ; 69(4): 264-270, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31323126

RESUMEN

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L.  monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L.  monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl-1 of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L.  monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L.  monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L.  monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L.  monocytogenes in wastewater from treatment plants. The LAMP assay detects L.  monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L.  monocytogenes detection.


Asunto(s)
Toxinas Bacterianas/genética , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Aguas Residuales/microbiología , Dominio AAA , Cartilla de ADN/genética , Humanos , Límite de Detección , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Purificación del Agua
19.
Future Microbiol ; 14: 801-828, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31271064

RESUMEN

Aim: Among the alternative sigma factors of Listeria monocytogenes, σB controls the largest regulon. The aim of this study was to perform a comprehensive review of σB-regulated genes, and the functions they confer. Materials & methods: A systematic search of PubMed and Web of Knowledge was carried out to identify members of the σB regulon based on experimental evidence of σB-dependent transcription and presence of a consensus σB-dependent promoter. Results: The literature review identified σB-dependent transcription units encompassing 304 genes encoding different functions including stress response and virulence. Conclusion: Our review supports the well-known roles of σB in virulence and stress response and provides new insight into novel roles for σB in metabolism and overall resilience of L. monocytogenes.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/genética , Metabolismo , Regulón , Factor sigma/metabolismo , Estrés Fisiológico , Listeria monocytogenes/patogenicidad , Listeria monocytogenes/fisiología , Virulencia
20.
J Food Sci ; 84(7): 1881-1887, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31264719

RESUMEN

Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real-time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real-time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre-enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50 ) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as "almost complete concordance" between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real-time PCR. PRACTICAL APPLICATION: The methodology described in this study significantly reduces the detection time of L. monocytogenes, when compared with culture-based methods, and it requires fewer steps than other molecular methods, making it a reliable and more convenient method for routine testing. Finally, the evaluation of the methodology in spiked food samples, confirms its reliability.


Asunto(s)
Productos Pesqueros/microbiología , Separación Inmunomagnética/métodos , Listeria monocytogenes/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Salmón/microbiología , Animales , Productos Pesqueros/análisis , Microbiología de Alimentos , Listeria monocytogenes/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinasas/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
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