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1.
Int J Food Microbiol ; 338: 108994, 2021 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-33279788

RESUMEN

The use of Whole genome sequencing (WGS) identified a multi-country outbreak of human listeriosis associated with consumption of frozen sweet corn produced in Hungary. The purpose of this report was to summarise information on the cases occurring in the UK which were part of this outbreak and outline investigations on the presence of Listeria monocytogenes in the affected food chain. Prior to the international recall of this product in 2018, 12 UK cases of listeriosis were identified as infected by the outbreak strain between 2015 and 18. Epidemiological and microbiological investigations confirmed these cases as belonging to the outbreak. A further case occurred in 2019 and a contaminated frozen pack from one of the implicated batches of sweet corn was recovered from the patient's domestic freezer. The outbreak strain was also detected in products from a sandwich manufacturer in 2018 which added frozen sweet corn directly to sandwich fillings. The sandwich manufacturer's sweet corn was supplied by a distributor in England which obtained frozen products from the Hungarian manufacturer implicated in the outbreak. Within the distributor's premises, 208 food and environmental samples were taken: L. monocytogenes was detected in 44% of 70 samples of frozen sweet corn and 5% of 79 other foods. The outbreak strain was detected in the frozen sweet corn, in one other frozen food (mixed vegetables) and in the factory environment. The outbreak strain was also recovered from frozen beans on retail sale in the first four months of 2019. Five other L. monocytogenes strains together with two other Listeria species were detected in samples from the importer's premises. One of the L. monocytogenes strains in the importer's factory, which was distinct from the outbreak strain, was also recovered from sweet corn collected from the sandwich manufacturer, sweet corn tested in England in 2013 and 2016 and the blood of two cases of human listeriosis which occurred in England in 2014. This report shows how analysis by WGS provides evidence to understand complex food chains. This report also highlights risks for transmission of human listeriosis from frozen sweet corn and the potential for misuse of this food as a ready-to-eat product.


Asunto(s)
Brotes de Enfermedades , Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos/microbiología , Listeriosis/epidemiología , Listeriosis/microbiología , Verduras/microbiología , Inglaterra , Enfermedades Transmitidas por los Alimentos/epidemiología , Congelación , Humanos , Listeria , Listeria monocytogenes/genética , Reino Unido , Secuenciación Completa del Genoma , Zea mays/microbiología
2.
Int J Food Microbiol ; 336: 108912, 2021 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-33091754

RESUMEN

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.


Asunto(s)
Antibacterianos/farmacología , Microbiología de Alimentos , Listeria monocytogenes , Carne de Cerdo/microbiología , Mataderos , Animales , Farmacorresistencia Bacteriana , Inocuidad de los Alimentos , Genotipo , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Serogrupo , Porcinos , Virulencia/genética
3.
Food Chem ; 338: 127837, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-32818863

RESUMEN

Early screening of L. monocytogenes in ready-to-eat food can prevent and control its harmful effects. In this study, we propose a highly sensitive magnetic DNA sensor based on nucleic acid hybridization reaction and magnetic signal readout. We design the L. monocytogenes specific probe1 and probe2 and label them on the 30 and 250 nm magnetic nanoparticles, respectively. The hybridization reaction between the magnetic probes and DNA of L. monocytogenes could form a sandwich nanocomplex. After magnetic separation, the unbound MNP30-probe2 can act as the transverse relaxation time (T2) signal readout probe. This assay allows the one-step detection of L. monocytogenes as low as 50 CFU/mL within 2 h without DNA amplification, and the average recovery in the spiked ham sausage samples can reach 92.6%. This system integrates the high sensitivity of magnetic sensing and high efficiency of hybridization reaction, providing a promising detection platform for pathogens.


Asunto(s)
Microbiología de Alimentos/métodos , Listeria monocytogenes , Productos de la Carne/microbiología , Hibridación de Ácido Nucleico/métodos , Sondas de ADN , ADN Bacteriano , Listeria monocytogenes/genética , Fenómenos Magnéticos , Espectroscopía de Resonancia Magnética , Nanopartículas/química , Técnicas de Amplificación de Ácido Nucleico
4.
Nat Commun ; 11(1): 6344, 2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33311493

RESUMEN

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillus probiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.


Asunto(s)
Lactobacillus casei/metabolismo , Listeria monocytogenes/efectos de los fármacos , Listeriosis/prevención & control , Probióticos/metabolismo , Probióticos/farmacología , Administración Oral , Animales , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Antígeno CD11c , Línea Celular , Chaperonina 60/metabolismo , Células Dendríticas , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Intestinos/microbiología , Células Asesinas Naturales , Lactobacillus casei/genética , Listeria/genética , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Ratones , Quinasa de Cadena Ligera de Miosina/metabolismo , FN-kappa B/metabolismo , Linfocitos T
5.
Sheng Wu Gong Cheng Xue Bao ; 36(11): 2334-2344, 2020 Nov 25.
Artículo en Chino | MEDLINE | ID: mdl-33244928

RESUMEN

Strain variability is one of the most important factors to influence the accuracy of foodborne pathogens risk assessment, such as Listeria monocytogenes, Salmonella spp. Strain-to-strain variation is defined as the inherent differences among identically treated strains of the same microbial species. The differences cannot be eliminated by changing test methods or improving test protocols. This review addresses presently related studies of strain variability. Based on the effect of strain variability on the outcome of risk assessment, we summarize sources of variabilities in food chain, strain phenotypic variabilities and the methods to integrate strain variability in growth and inactivation into predictive modelling, and indicate the inadequacies in the study of strain variability. We suggest further study the mechanism of strain variability, expand the comparison of variability among different sources, and integrate the variability of gene expression, protein and cell metabolism into the predictive modelling.


Asunto(s)
Microbiología de Alimentos , Listeria monocytogenes , Listeria monocytogenes/genética , Medición de Riesgo , Salmonella/genética
6.
BMC Infect Dis ; 20(1): 721, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-33004020

RESUMEN

BACKGROUND: Listeria monocytogenes (L. monocytogenes) is a facultative intracellular bacterial pathogen which can invade different mammalian cells and reach to the central nervous system (CNS), leading to meningoencephalitis and brain abscesses. In the diagnosis of L. monocytogenes meningoencephalitis (LMM), the traditional test often reports negative owing to the antibiotic treatment or a low number of bacteria in the cerebrospinal fluid. To date, timely diagnosis and accurate treatment remains a challenge for patients with listeria infections. CASE PRESENTATION: We present the case of a 66-year-old woman whose clinical manifestations were suspected as tuberculous meningoencephalitis, but the case was finally properly diagnosed as LMM by next-generation sequencing (NGS). The patient was successfully treated using a combined antibacterial therapy, comprising ampicillin and trimethoprim-sulfamethoxazole. CONCLUSION: To improve the sensitivity of LMM diagnosis, we used NGS for the detection of L. monocytogenes. Hence, the clinical utility of this approach can be very helpful since it provides quickly and trust results.


Asunto(s)
Listeria monocytogenes/genética , Meningitis por Listeria/microbiología , Meningoencefalitis/microbiología , Anciano , Ampicilina/uso terapéutico , Antibacterianos/uso terapéutico , Absceso Encefálico/tratamiento farmacológico , Errores Diagnósticos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Listeria monocytogenes/aislamiento & purificación , Meningitis por Listeria/diagnóstico , Meningitis por Listeria/tratamiento farmacológico , Meningoencefalitis/diagnóstico , Meningoencefalitis/tratamiento farmacológico , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Tuberculosis Meníngea/diagnóstico , Tuberculosis Meníngea/microbiología
7.
Appl Environ Microbiol ; 86(22)2020 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-32887717

RESUMEN

Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages.IMPORTANCE Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.


Asunto(s)
Bacteriófagos/fisiología , Especificidad del Huésped , Listeria monocytogenes/genética , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/prevención & control , Listeria monocytogenes/virología , Listeriosis/microbiología , Listeriosis/prevención & control , Mutación
8.
Int J Food Microbiol ; 335: 108885, 2020 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-32947145

RESUMEN

Worldwide, Listeria monocytogenes is a common foodborne pathogen and serotyping is necessary for traceability and surveillance. Due to high accuracy and speed, PCR is commonly used for serotyping by targeting specific genes, such as lmo1118 and ORF2110 for 1/2c-3c and 4b-4d-4e, respectively. Single nucleotide polymorphisms (SNPs) are single base alterations at specific loci which are associated with various phenomena. In this study, a method was explored to mine specific SNPs from 253 L. monocytogenes genomes with known serotypes by writing Python programming language script. After blasting all the CDS, seventeen candidate genes with specific SNPs were obtained and these genes contained multiple types of SNPs, including lineages I, II, III, 1/2a-3a, 1/2b-3b-7, and 1/2c-3c specific SNPs. The sensitivity and specificity of these candidate SNP sites are higher than 85% in the genomes examined. Combining lineage I-specific, lineage II-specific, 1/2b-3b-7-specific, and 1/2c-3c-specific SNP sites together, using allele-specific multiplex PCR, we could serotype major L. monocytogenes serotypes. This method was used for 60 L. monocytogenes strains isolated from various food samples and the serotyping results were 100% identical to those of the currently accepted method with the reduced primers from ten to eight. Our results indicate that allele-specific multiplex PCR after mining specific SNPs from common genome database can be used for bacterial typing.


Asunto(s)
Microbiología de Alimentos/métodos , Listeria monocytogenes/aislamiento & purificación , Listeriosis/diagnóstico , Polimorfismo de Nucleótido Simple/genética , Serotipificación/métodos , Alelos , Humanos , Listeria monocytogenes/genética , Reacción en Cadena de la Polimerasa Multiplex , Sensibilidad y Especificidad , Serogrupo
9.
Proc Natl Acad Sci U S A ; 117(38): 23774-23781, 2020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32878997

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Listeria monocytogenes , ARN Bacteriano/química , ARN Mensajero/química , Regiones no Traducidas 5'/genética , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Replicación del ADN/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/patogenicidad , Listeriosis , Ratones , Conformación de Ácido Nucleico , ARN Bacteriano/genética , ARN Mensajero/genética
10.
Int J Food Microbiol ; 334: 108801, 2020 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-32795712

RESUMEN

In the summer of 2014, a multistate outbreak of listeriosis associated with contaminated stone fruit (peach and nectarine) was reported. A serotype 4b variant Listeria monocytogenes (Lm) strain of singleton Sequence Type (ST) 382 was isolated from clinical samples and stone fruit associated with the outbreak. A serotype 1/2b Lm strain of ST5, Clonal Complex 5 was isolated only from outbreak-associated stone fruit, not from clinical samples. Here we investigated the fate of the serotype 4b and 1/2b strains, at two inoculation levels (high level at 3.7 logCFU/fruit and low level at 2.7 logCFU/fruit), on the surfaces of white peach, yellow peach and yellow nectarine stored at 4 °C for 26 days. After rinsing the fruits, we determined the Lm levels in the rinsates and on the peels. We enumerated Lm using a direct plating method and compared two chromogenic agars. The Lm populations rapidly declined in the first 3 days and then declined more slowly until Day 19/21. The maximum decline was 1.6 logCFU/fruit on yellow peach inoculated with serotype 4b at high level. For fruits inoculated with high-level Lm, the lowest level of Lm (1.7 logCFU/fruit) was observed on for white peach inoculated with serotype 1/2b, and the highest level of Lm (2.6 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with the serotype 1/2b strain. For fruits inoculated with low-level Lm, the lowest level of Lm (1.3 logCFU/fruit) was observed on yellow nectarine inoculated with either the serotype 4b or 1/2b strain, and the highest level of Lm (1.7 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with ST382. The D-values ranged from 15 days to 28 days. Lm remained viable until the end of storage (Day 26), but the levels were not significantly different from those on Day 19/21. The types of stone fruit and Lm strain did not significantly affect the survival of Lm. These results demonstrate that contaminated stone fruit can carry a potential risk for causing listeriosis in susceptible populations. Comparison of direct plating results using two chromogenic agars showed that RAPID' L. mono and Agar Listeria Ottavani & Agosti performed equivalently for enumerating Lm on stone fruit. The fruit rinsing recovered 80% to 84% of Lm from fruit surfaces.


Asunto(s)
Brotes de Enfermedades , Frutas/microbiología , Listeria monocytogenes/fisiología , Listeriosis/microbiología , Prunus persica/microbiología , Frío , Microbiología de Alimentos , Frutas/clasificación , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/aislamiento & purificación , Listeriosis/epidemiología , Viabilidad Microbiana , Prunus persica/clasificación , Serogrupo
11.
J Food Sci ; 85(9): 2889-2895, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32794185

RESUMEN

This study was aimed to investigate the presence of Listeria monocytogenes in raw water buffalo milk and milk products, besides determining its serotype and the extent of its resistance against various antibiotics. A total of 188 samples of raw water buffalo milk and milk products were collected from Samsun Province, Turkey between November 2012 and May 2013. The classical culture technique was used to isolate and identify L. monocytogenes, as described in EN ISO 11290-1. The isolates were confirmed as L. monocytogenes by using PCR with (hylA) primers specific for the hemolysin gene. The antimicrobial susceptibility test was achieved by using the VITEK 2 compact system and VITEK 2 AST-P640 card. L. monocytogenes was found in 7 (3.7%) of the 188 samples. Four of them were obtained from cheese and three from milk samples. Whereas, L. monocytogenes was not detected in any of the clotted cream samples. A total of 13 isolates were confirmed by PCR as L. monocytogenes. Among these isolates, one was 1/2c (or 3c) (7.6%), three were 4b (or 4d, 4e) (23%), four were 1/2b (or 3b) (30.7%), and the other five isolates were serotype 1/2a (or 3a) (38.4%). The highest antimicrobial resistance was recorded against fosfomycine (100%) followed by oxacillin (92%), penicillin (84%), and erythromycin (69%). However, no resistance was determined against ciprofloxacin, gentamicin, and tigecycline. PRACTICAL APPLICATION: This study showed that some samples of raw buffalo milk and the milk products were contaminated with Listeria monocytogenes. The serotype with the highest prevalence was determined as L. monocytogenes 1/2a. This study also demonstrated that most of the L. monocytogenes isolates had developed multiresistance to many frequently used medical antimicrobial agents.


Asunto(s)
Queso/microbiología , Farmacorresistencia Bacteriana , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/aislamiento & purificación , Leche/microbiología , Animales , Antibacterianos/farmacología , Búfalos , Cartilla de ADN/genética , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Reacción en Cadena de la Polimerasa , Serotipificación , Turquia
12.
PLoS One ; 15(7): e0233945, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32701964

RESUMEN

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.


Asunto(s)
Brevibacterium/metabolismo , Queso/microbiología , Etanolamina/metabolismo , Microbiología de Alimentos , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/genética , Propilenglicol/metabolismo , Psychrobacter/metabolismo , Transcriptoma , Aclimatación , Agar , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Técnicas de Cocultivo , Medios de Cultivo , Transporte de Electrón/genética , Fermentación/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidad , Plásmidos , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Pequeño no Traducido/biosíntesis , ARN Pequeño no Traducido/genética , Virulencia/genética
13.
Appl Environ Microbiol ; 86(17)2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32591377

RESUMEN

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.


Asunto(s)
Fenómenos Fisiológicos Bacterianos , Regulación Bacteriana de la Expresión Génica , Aptitud Genética , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Listeria monocytogenes/crecimiento & desarrollo , Especificidad de la Especie , Transcripción Genética , Virulencia/genética
14.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-32414794

RESUMEN

Listeria monocytogenes is a pathogen mostly associated with the consumption of ready-to-eat foods and can cause severe disease and death. It can be introduced into food chains from raw materials, but often the contamination source is the food production environment, where certain clones can persist for years. In the meat chain, ST9 is one of the most commonly encountered L. monocytogenes sequence types, and for effective source tracking, the divergence and spread of ST9 must be understood. In this study, whole-genome sequencing (WGS) was used to characterize and track 252 L. monocytogenes ST9 isolates collected from four Norwegian meat processing plants between 2009 and 2017. The isolates formed distinct clusters relative to genomes found in public databases, and all but three isolates clustered into two major clonal populations. Different contamination patterns were revealed, e.g., evidence of contamination of two factories with a clone that diverged from its ancestor in the late 1990s through a common source of raw materials; breach of hygienic barriers within a factory, leading to repeated detection of two clones in the high-risk zone during a 4- to 6-year period; entry through the purchase and installation of second-hand equipment harboring a previously established clonal population; and spreading and diversification of two clones from two reservoirs within the same production room over a 9-year period. The present work provides data on the diversity of ST9, which is crucial for epidemiological investigations and highlights how WGS can be used for source tracking within food processing factories.IMPORTANCE Listeria monocytogenes is a deadly foodborne pathogen that is widespread in the environment, and certain types can be established in food factories. The sequence type ST9 dominates in meat processing environments, and this work was undertaken to obtain data needed for the tracking of this subtype. By using whole-genome sequencing (WGS), we revealed the presence of cross-contamination routes between meat factories as well as within a single factory, including the spread from different reservoirs within the same room. It was also possible to estimate the time frame of persistence in the factory, as well as when and how new clones had entered. The present work contributes valuable information about the diversity of ST9 and exemplifies the potential power of WGS in food safety management, allowing the determination of relationships between strains both in an international context and locally between and within factories.


Asunto(s)
Microbiología de Alimentos , Variación Genética , Listeria monocytogenes/genética , Listeriosis/transmisión , Industria para Empaquetado de Carne , Carne/microbiología , Inocuidad de los Alimentos , Tipificación de Secuencias Multilocus , Noruega , Polimorfismo de Nucleótido Simple , Secuenciación Completa del Genoma
15.
Int J Food Microbiol ; 324: 108614, 2020 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-32371237

RESUMEN

Organic acids such as fumarate are commonly used as antimicrobials in foods. Apart from the classical mechanism of intracellular dissociation, weak acids are active through important additional mechanisms which are not well-defined. Fumarate, based on its low dissociation constants is expected to have a low antimicrobial activity which is not the case, suggesting additional antimicrobial effects. Previously, fumarate has been shown to inhibit the GAD system of E. coli and therefore, we investigated for first time how it affects this system in Listeria monocytogenes. We found that fumarate is highly antimicrobial towards L. monocytogenes under acidic conditions. We also show that in cell lysates and similarly to E. coli, fumarate inhibits the GAD system of L. monocytogenes. However, despite the inhibition and in contrast to E. coli, L. monocytogenes is able to counteract this and achieve a higher extracellular GAD output (measured by GABA export) in the presence of fumarate compared to its absence. The latter is achieved by a dramatic 9.44-fold increase in the transcription of gadD2 which is the main component of the extracellular GAD system. Interestingly, although maleate, the cis-isomer of fumarate results in a more dramatic 48.5-fold gadD2 upregulation than that of fumarate, the final GADe output is lower suggesting that maleate might be a stronger inhibitor of the GAD system. In contrast, the GADe removes more protons in the presence of fumarate than in the presence of HCl at the same pH. All the above suggest that there are additional effects by fumarate which might be associated with the intracellular GAD system (GADi) or other acid resistance systems. We assessed the GADi output by looking at the intracellular GABA pools which were not affected by fumarate. However, there are multiple pathways (e.g. GABA shunt) that can affect GABAi pools and we cannot conclusively suggest that GADi is affected. Furthermore, similarly to maleate, fumarate is able to eliminate L. monocytogenes in biofilms under acidic conditions. Overall, fumarate is a good candidate for L. monocytogenes decontamination and biofilm removal which is not toxic compared to the toxic maleate.


Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Fumaratos/farmacología , Glutamato Descarboxilasa/antagonistas & inhibidores , Listeria monocytogenes/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Concentración de Iones de Hidrógeno , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Maleatos/farmacología
16.
PLoS One ; 15(4): e0231393, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32352974

RESUMEN

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.


Asunto(s)
Microbiología de Alimentos , Variación Genética , Listeria monocytogenes/genética , Virulencia/genética , Proteínas Bacterianas/genética , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Humanos , Listeria monocytogenes/clasificación , Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/patogenicidad , Listeriosis/patología , Listeriosis/transmisión , Tipificación de Secuencias Multilocus , Filogenia , Plásmidos/genética , Plásmidos/metabolismo , Serogrupo , Secuenciación Completa del Genoma
17.
An Acad Bras Cienc ; 92 Suppl 1: e20180557, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32348408

RESUMEN

In Brazil and in other countries of the world, studies have been conducted to identify Listeria monocytogenes in cattle meat that is preferably consumed undercooked and, when marketed without meeting strict phytosanitary requirements, may cause outbreaks of listeriosis. In the such, foodborne outbreaks, the methods used for the detection of the pathogen and the efficiency associated with them are crucial for the proper assessment. In this study, we used the techniques biochemical and molecular for identification of the L. monocytogenes isolated from 30 samples of the fresh beef, marketed in ten butchers' shop of the free-fair from a municipality from the Bahia, Brazil. The results obtained from biochemical tests (catalase, motility, ß-hemolysis and carbohydrate fermentation), as well as PCR analysis for the hly gene (hemolysin production is an important factor in the pathogenesis of listeriosis) revealed that 50% of butchers shops presented bovine meat contaminated with bacteria of the Listeria sp. and confirmed that 54.16% of the analyzed meat samples were positive for L. monocytogenes. This study highlights the importance of microbiological surveillance in free-fair to minimize the exposure of consumers to this foodborne pathogen.


Asunto(s)
ADN Bacteriano/análisis , Proteínas Hemolisinas/genética , Listeria monocytogenes/aislamiento & purificación , Productos de la Carne/microbiología , Animales , Brasil , Bovinos , Microbiología de Alimentos , Proteínas Hemolisinas/análisis , Listeria monocytogenes/genética
18.
Food Microbiol ; 90: 103481, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32336364

RESUMEN

The Glutamate Decarboxylase (GAD) system is important for survival of L. monocytogenes and other microorganisms under acidic conditions. Environmental conditions influence the function of the GAD system. Until now, the only conditions known to lead to increased transcription of the GAD system are the stationary phase in rich media and anoxic conditions. Previously, we showed that transcription of the GAD system requires unidentified compounds other than glutamate present in rich media. Following a test looking at various compounds we identified for first time that peptone, tryptone and casamino acids activate the GAD system under oxic conditions suggesting that amino acid(s) other than glutamate and/or peptides are important for the above process. The defined medium, where the GAD system is inactive, once it is supplemented with the above compounds results in an active intracellular and extracellular GAD system and increased acid resistance. Through functional genomics we show that these compounds are required for GadD2 activity and although we previously showed that GadD3 is active part of the intracellular GAD system, the supplementation did not activate this gene. The above is explained by the fact that only gadD2 transcription was upregulated by these compounds while the transcription of gadD1 and gadD3 remained unaffected. Together our results show that the L. monocytogenes GadD2 decarboxylase is activated in the presence of amino acids or peptides other than glutamate, a finding that has important implications for acid tolerance and food safety.


Asunto(s)
Ácidos/metabolismo , Aminoácidos/metabolismo , Glutamato Descarboxilasa/genética , Ácido Glutámico/metabolismo , Listeria monocytogenes/enzimología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Listeria monocytogenes/genética
19.
PLoS Pathog ; 16(4): e1008446, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32282860

RESUMEN

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.


Asunto(s)
Disentería Bacilar/microbiología , Enterocitos/microbiología , Células Epiteliales/microbiología , Listeria monocytogenes/fisiología , Listeriosis/microbiología , Shigella flexneri/fisiología , Células CACO-2 , Humanos , Listeria monocytogenes/genética , Shigella flexneri/genética
20.
Biochemistry ; 59(10): 1124-1136, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32125848

RESUMEN

ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.


Asunto(s)
Corrinoides/metabolismo , Listeria monocytogenes/enzimología , Aciltransferasas/metabolismo , Adenosina Trifosfato/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Aldehído Oxidorreductasas/ultraestructura , Transferasas Alquil y Aril/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Catálisis , Dominio Catalítico , Cobalto/química , Cobamidas/metabolismo , Cinética , Lactobacillus reuteri/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Modelos Moleculares , Mutación , Transferasas/metabolismo
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