Dataset Information

Characteristics for phage-encoded cell wall hydrolase of LysSAP27 to reduce staphylococcal food poisoning.

ABSTRACT: To reduce staphylococcal food poisoning (SFP), a phage-encoded cell wall hydrolase called endolysin has emerged as an attractive antibacterial agent. In this study, the Staphylococcus aureus infecting phage vB_SauS-SAP27 (ϕSAP27) was isolated from sewage and characterized morphologically and genetically. ϕSAP27 was identified as Siphoviridae temperate phage, with a genome of 43 kbp. A ϕSAP27 endolysin named LysSAP27 was produced recombinantly in Escherichia coli. LysSAP27 exhibited the highest activity at neutral pH and a temperature of 30 °C, and its lytic activity was upregulated by calcium ions. Following optimization of the enzymatic conditions, LysSAP27 was applied to S. aureus-contaminated milk. Treatment with 2 μM LysSAP27 led to a significant bactericidal effect, corresponding to a reduction in bacterial titer by 2.8 log CFU/mL within 1 h and 3.4 log CFU/mL within 2 h. Therefore, LysSAP27 could be used as an effective antimicrobial agent to prevent SFP in food.

Supplementary information

The online version contains supplementary material available at 10.1007/s10068-021-00910-2.

SUBMITTER: Park DW

PROVIDER: S-EPMC8144258 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Characteristics for phage-encoded cell wall hydrolase of LysSAP27 to reduce staphylococcal food poisoning.

Park Do-Won DW Lee Young-Duck YD Park Jong-Hyun JH

Food science and biotechnology 20210511 5

To reduce staphylococcal food poisoning (SFP), a phage-encoded cell wall hydrolase called endolysin has emerged as an attractive antibacterial agent. In this study, the Staphylococcus aureus infecting phage vB_SauS-SAP27 (ϕSAP27) was isolated from sewage and characterized morphologically and genetically. ϕSAP27 was identified as Siphoviridae temperate phage, with a genome of 43 kbp. A ϕSAP27 endolysin named LysSAP27 was produced recombinantly in Escherichia coli. LysSAP27 ex ...[more]

PMID: 34123470

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Project description:Through staphylococcal enterotoxin (SE) production, Staphylococcus aureus is a common cause of food poisoning. Detection of staphylococcal food poisoning (SFP) is mostly performed using immunoassays, which, however, only detect five of 27 SEs described to date. Polymerase chain reactions are, therefore, frequently used in complement to identify a bigger arsenal of SE at the gene level (se) but are labor-intensive. Complete se profiling of isolates from different sources, i.e., food and human cases, is, however, important to provide an indication of their potential link within foodborne outbreak investigation. In addition to complete se gene profiling, relatedness between isolates is determined with more certainty using pulsed-field gel electrophoresis, Staphylococcus protein A gene typing and other methods, but these are shown to lack resolution. We evaluated how whole genome sequencing (WGS) can offer a solution to these shortcomings. By WGS analysis of a selection of S. aureus isolates, including some belonging to a confirmed foodborne outbreak, its added value as the ultimate multiplexing method was demonstrated. In contrast to PCR-based se gene detection for which primers are sometimes shown to be non-specific, WGS enabled complete se gene profiling with high performance, provided that a database containing reference sequences for all se genes was constructed and employed. The custom compiled database and applied parameters were made publicly available in an online user-friendly interface. As an all-in-one approach with high resolution, WGS additionally allowed inferring correct isolate relationships. The different DNA extraction kits that were tested affected neither se gene profiling nor relatedness determination, which is interesting for data sharing during SFP outbreak investigation. Although confirming the production of enterotoxins remains important for SFP investigation, we delivered a proof-of-concept that WGS is a valid alternative and/or complementary tool for outbreak investigation.

Project description:Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP.ImportanceThe importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Project description:BackgroundCharacteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants.ResultsThree MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings.ConclusionThis study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

Dataset Information

Characteristics for phage-encoded cell wall hydrolase of LysSAP27 to reduce staphylococcal food poisoning.

Supplementary information

Publications

Characteristics for phage-encoded cell wall hydrolase of LysSAP27 to reduce staphylococcal food poisoning.

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