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Bio-protocol Feb 2022Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a...
Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution. Graphic abstract: Engineering to express NanoLuc and its application in bioluminescence assay.
PubMed: 35284607
DOI: 10.21769/BioProtoc.4308 -
Emerging Microbes & Infections Dec 2021is an important foodborne pathogen, and is ubiquitously distributed in the natural environment. Cattle and sheep, as natural hosts, can transmit to related meat and...
is an important foodborne pathogen, and is ubiquitously distributed in the natural environment. Cattle and sheep, as natural hosts, can transmit to related meat and dairy products. In this study, the prevalence, distribution, and transmission characteristics of were analysed by investigating 5214 samples of cattle and sheep in farm and slaughtering environments in China. A low contamination incidence of (0.5%, 20/4430) was observed in farm environment, but there was a high contamination incidence in slaughtering environment (9.4%, 74/784). The incidence of in cattle and sheep farm and slaughtering environments is more common and significantly higher (9.7%, 508/5214) than that of (1.8%, 94/5214). The distinct molecular and genetic characteristics of by PFGE and MLST indicated that and were gradually transmitted from the farm and slaughtering environments to end products, such as beef and mutton along the slaughtering chain. The ST7, ST9, ST91, and ST155 found in our study were associated with the human listeriosis cases in China. In addition, the findings of virulence markers (, , LIPI-3 LIPI-4, and ECIII) concerned with the pathogenesis of human listeriosis and antibiotics resistance of in this study implies a potential public health risk. This study fills the gap in the epidemiology of beef cattle and sheep that carry in farm and slaughtering environments in major cattle and sheep producing areas in China.
Topics: Abattoirs; Animals; Cattle; Cattle Diseases; China; Farms; Food Handling; Food Safety; Listeria; Listeriosis; Meat; Prevalence; Sheep; Sheep Diseases
PubMed: 33560938
DOI: 10.1080/22221751.2021.1888658 -
Microbial Genomics Jul 2022() is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular...
() is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross-protective barriers, including the placenta. One family of virulence factors, internalins, contributes to pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains that enable protein-protein interactions, allowing to bind host proteins. Notably, other species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus . Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Although prior studies of InlP have begun to elucidate its role in pathogenesis, there remains a lack of information regarding homologs in other species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional species. We found that () and () encode InlP homologs. We also found InlP-like homologs in and the recently identified species . All newly identified homologs lack the full-length LRR6 and LRR7 domains found in 's InlP. These findings are informative regarding the evolution of one key virulence factor, InlP, and serve as a springboard for future evolutionary studies of pathogenesis as well as mechanistic studies of internalins.
Topics: Bacterial Proteins; Female; Humans; Listeria; Listeria monocytogenes; Listeriosis; Placenta; Pregnancy; Virulence Factors
PubMed: 35904424
DOI: 10.1099/mgen.0.000828 -
Carbohydrate Research Jan 2022Listeria innocua is genetically closely related to the foodborne human pathogen Listeria monocytogenes. However, as most L. innocua strains are non-pathogenic, it has...
Listeria innocua is genetically closely related to the foodborne human pathogen Listeria monocytogenes. However, as most L. innocua strains are non-pathogenic, it has been proposed as a surrogate organism for determining the efficacy of antimicrobial strategies against L. monocytogenes. Teichoic acids are one of the three major cell wall components of Listeria, along with the peptidoglycan backbone and cell wall-associated proteins. The polymeric teichoic acids make up the majority of cell wall carbohydrates; the type of teichoic acids directly attached to the peptidoglycan are termed wall teichoic acids (WTAs). WTAs play vital physiological roles, are important virulence factors, antigenic determinants, and phage-binding ligands. The structures of the various WTAs of L. monocytogenes are well known, whereas those of L. innocua are not. In the present study, the WTA structure of L. innocua ŽM39 was determined mainly by 1D and 2D NMR spectroscopy and it was found to be the following: [→4)-[α-D-GlcpNAc-(1→3)]-β-D-GlcpNAc-(1→4)-D-Rbo-(1P→] This structure is new with respect to all currently known Listeria WTAs and it shares structural similarities with type II WTA serovar 6a. In addition, the genome of strain L. innocua ŽM39 was sequenced and the majority of putative WTA synthesis genes were identified.
Topics: Cell Wall; Humans; Listeria; Listeria monocytogenes; Teichoic Acids
PubMed: 35007911
DOI: 10.1016/j.carres.2021.108499 -
Poultry Science Sep 2012Listeria monocytogenes is a ubiquitous, saprophytic, Gram-positive bacterium and occasional food-borne pathogen, often associated with ready-to-eat meat products....
Listeria monocytogenes is a ubiquitous, saprophytic, Gram-positive bacterium and occasional food-borne pathogen, often associated with ready-to-eat meat products. Because of the increased consumer interest in organic, all natural, and free range poultry products, it is important to understand L. monocytogenes in the context of such systems. Pasture-reared poultry were surveyed over the course of two 8-wk rearing periods. Cecal, soil, and grass samples were collected for Listeria isolation and characterization. Seven of 399 cecal samples (or 1.75%) were Listeria-positive. All positive cecal samples were obtained from broilers sampled at 2 wk of age. Grass and soil samples were collected from the pasture both before and after introduction of the poultry. Environmental samples collected after introduction of poultry were significantly more likely to contain Listeria (P < 0.001). The results of analytical profile index Listeria, sigB allelic typing, and hlyA PCR tests found that both L. monocytogenes and L. innocua, including hemolytic L. innocua, were recovered from the cecal and environmental (grass/soil) samples. The sigB allelic typing also revealed that (1) positive samples could be composed of 2 or more allelic types; (2) allelic types found in cecal samples could also be found in the environment; and (3) allelic types could persist through the 2 rearing periods. Our data indicate that both pasture-reared poultry and their environment can be contaminated with L. monocytogenes and hemolytic L. innocua.
Topics: Animal Husbandry; Animals; Cecum; Chickens; Housing, Animal; Listeria; Listeriosis; Phylogeny; Poaceae; Poultry Diseases; Soil Microbiology
PubMed: 22912449
DOI: 10.3382/ps.2012-02292 -
Microorganisms Feb 2020The genus now comprises up to now 21 recognized species and six subspecies, with and as the most prevalent sensu stricto associated species. Reports focusing on the...
The genus now comprises up to now 21 recognized species and six subspecies, with and as the most prevalent sensu stricto associated species. Reports focusing on the challenges in detection and confirmation are available, especially from food-associated environmental samples. is more prevalent in the food processing environment (FPE) than and has been shown to have a growth advantage in selective enrichment and agar media. Until now, the adaptive nature of L. innocua in FPEs has not been fully elucidated and potential persistence in the FPE has not been observed. Therefore, the aim of this study is to characterize (n = 139) and (n = 81) isolated from FPEs and cheese products collected at five dairy processing facilities (A-E) at geno- and phenotypic levels. Biochemical profiling was conducted for all and the majority of (n = 124) isolates and included a rhamnose positive reaction. isolates were most frequently confirmed as PCR-serogroups 1/2a, 3a (95%). Pulsed-field gel electrophoresis (PFGE)-typing, applying the restriction enzymes AscI, revealed 33 distinct Listeria PFGE profiles with a Simpson's Index of Diversity of 0.75. Multi-locus sequence typing (MLST) resulted in 27 STs with seven new L. innocua local STs (ST1595 to ST1601). ST1597 and ST603 and ST121 and ST14 were the most abundant genotypes in dairy processing facilities A-E over time. Either SSI-1 (ST14) or SSI-2 (ST121, all L. innocua) were present in successfully FPE-adapted strains. We identified housekeeping genes common in Listeria isolates and L. monocytogenes genetic lineage III. Wherever there are long-term contamination events of L. monocytogenes and other Listeria species, subtyping methods are helpful tools to identify niches of high risk.
PubMed: 32050536
DOI: 10.3390/microorganisms8020234 -
Frontiers in Microbiology 2023The similarity of the genome with and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible...
The similarity of the genome with and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an in-depth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, X, in the isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of . The five isolates possessed an ATP-dependent protease (L) gene, which mediates heat resistance, on a 25 type plasmids. Blast analysis of L-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to that was responsible for a serious outbreak, this is the first report of containing L-carrying plasmids. Various genetic mechanisms of virulence transfer among species and other genera could raise the possibility of the evolution of virulent strains of . Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures.
PubMed: 37234542
DOI: 10.3389/fmicb.2023.1160244 -
Frontiers in Microbiology 2021Studies of biofilm formation by bacteria are crucial for understanding bacterial resistance and for development of novel antibacterial strategies. We have developed a...
Studies of biofilm formation by bacteria are crucial for understanding bacterial resistance and for development of novel antibacterial strategies. We have developed a new bioluminescence biofilm assay for , which is considered a non-pathogenic surrogate for was transformed with a plasmid for inducible expression of NanoLuc luciferase (Nluc). Concentration-dependent bioluminescence signals were obtained over a concentration range of more than three log units. This biofilm assay enables absolute quantification of bacterial cells, with the necessary validation. For biofilm detection and quantification, this "Nluc bioluminescence" method has sensitivity of 1.0 × 10 and 3.0 × 10 colony forming units (CFU)/mL, respectively, with a dynamic range of 1.0 × 10 to 5.0 × 10 CFU/mL. These are accompanied by good precision (coefficient of variation, <8%) and acceptable accuracy (relative error for most samples, <15%). This novel method was applied to assess temporal biofilm formation of as a function of concentration of inoculant, in comparison with conventional plating and CFU counting, the crystal violet assay, and the resazurin fluorescence assay. Good correlation ( = 0.9684) of this Nluc bioluminescence assay was obtained with CFU counting. The limitations of this Nluc bioluminescence assay include genetic engineering of bacteria and relatively high cost, while the advantages include direct detection, absolute cell quantification, broad dynamic range, low time requirement, and high sensitivity. Nluc-based detection of should therefore be considered as a viable alternative or a complement to existing methods.
PubMed: 33633716
DOI: 10.3389/fmicb.2021.636421 -
Infection and Immunity Apr 2019is considered a nonpathogenic species. Natural atypical hemolytic isolates have been reported but have not been characterized in detail. Here, we report the genomic... (Comparative Study)
Comparative Study
is considered a nonpathogenic species. Natural atypical hemolytic isolates have been reported but have not been characterized in detail. Here, we report the genomic and functional characterization of representative isolates from the two known natural hemolytic clades. Whole-genome sequencing confirmed the presence of pathogenicity islands (LIPI) characteristic of species. Functional assays showed that LIPI-1 and genes are transcribed, and the corresponding gene products are expressed and functional. Using and assays, we show that atypical hemolytic is virulent, can actively cross the intestinal epithelium, and spreads systemically to the liver and spleen, albeit to a lesser degree than the reference EGDe strain. Although human exposure to hemolytic is likely rare, these findings are important for food safety and public health. The presence of virulence traits in some clades supports the existence of a common virulent ancestor of and .
Topics: Animals; Bacterial Proteins; Bird Diseases; Ducks; Feces; Galliformes; Genome, Bacterial; Genomic Islands; Humans; Listeria; Listeria monocytogenes; Listeriosis; Phylogeny; Serotyping; Virulence; Whole Genome Sequencing
PubMed: 30670551
DOI: 10.1128/IAI.00758-18 -
Foods (Basel, Switzerland) Aug 2023The antibacterial effect of pomegranate juice (PJ) at six concentrations (0, 10, 20, 30, 40, and 50%) against and was investigated in distilled water (DW) and...
The antibacterial effect of pomegranate juice (PJ) at six concentrations (0, 10, 20, 30, 40, and 50%) against and was investigated in distilled water (DW) and bacterial culture broth. and at approximately 10 cfu mL were inoculated in PJ samples and incubated at 4, 25, and 37 °C for 0, 6, 24, and 48 h. The bacterial population and pH of culture media were measured at each removal. Results indicated that the antibacterial effect of PJ was dependent upon bacteria species, juice concentration, incubation temperature, and growth medium. Higher juice concentration and incubation temperature resulted in increased antibacterial effects. Bacterial populations were decreased more significantly in DW systems than in the culture broth, while was more sensitive to PJ than in the DW systems. Regardless of PJ concentrations in DW systems, initially inoculated at approximately 10 cfu mL, was reduced to undetectable levels at 25 and 37 °C within 24 h. The growth of and was significantly inhibited in bacterial culture broth containing ≥ 20% PJ ( < 0.001). This study provides insight into the potential application of PJ in food and beverage products for food protection.
PubMed: 37685180
DOI: 10.3390/foods12173247