Discovery and characterization of a new genotype of Salmonella enterica serovar Bareilly isolated from diarrhea patients of food-borne outbreaks.

Since the first food-borne outbreak of Salmonella enterica serovar Bareilly in the UK (2010), it has been recognized as a new type of food-borne pathogen in S. enterica. To detect and characterize this new serovar pathogen in South Korea, a total of 175 Salmonella strains was isolated and 31 isolates were identified as S. Bareilly from various food-borne outbreaks between 2014 and 2018. While pulsed-field gel electrophoresis (PFGE) analysis using XbaI revealed two major groups (A and B) each with two subgroups (A1, A2/B1, B2), average nucleotide identity (ANI), single nucleotide polymorphism (SNP), and in silico multilocus sequence typing (MLST) analyses confirmed only two major groups. Interestingly, extended SNP analysis with 67 S. Bareilly strains from outbreaks in other countries revealed that A group strains between 2014 and 2016 shared a close evolutionary relationship with the strains from outside of South Korea; however, the B group strains in 2018 were located in a separate SNP tree branch. These findings suggest that the A group may share common ancestor with the strains of previous outbreaks in the UK or other countries, while the B group is a new genotype. Comparative virulence factor (VF) analysis between the A and B group strains showed that S. Bareilly in the B group has more various than that of the A group. A comparative biofilm formation assay supports for this, which B group strain GG-21 has higher biofilm formation activity than A group strain GG-07. Antibiotic susceptibility test of 31 S. Bareilly strains revealed high susceptibility to 17 tested antibiotics, suggesting that S. Bareilly can be easily treated by antibiotics.

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain.

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

The Novel Enterococcus Phage vB_EfaS_HEf13 Has Broad Lytic Activity Against Clinical Isolates of Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive, facultative anaerobic bacterium frequently found in the gastrointestinal tract, oral cavity, and periodontal tissue. Although it is considered a commensal, it can cause bacteremia, endocarditis, endodontic infections, and urinary tract infections. Because antibiotics are cytotoxic not only to pathogens, but also to health-beneficial commensals, phage therapy has emerged as an alternative strategy to specifically control pathogenic bacteria with minimal damage to the normal flora. In this study, we isolated a novel phage, Enterococcus phage vB_EfaS_HEf13 (phage HEf13), with broad lytic activity against 12 strains of E. faecalis among the three laboratory strains and 14 clinical isolates of E. faecalis evaluated. Transmission electron microscopy showed that phage HEf13 has morphological characteristics of the family Siphoviridae. Phage HEf13 was stable at a wide range of temperature (4-60°C) and showed tolerance to acid or alkaline (pH 3-12) growth conditions. Phage HEf13 had a short latent period (25 min) with a large burst size (approximately 352 virions per infected cell). The lytic activity of phage HEf13 at various multiplicities of infection consistently inhibited the growth of diverse clinical isolates of E. faecalis without any lysogenic process. Moreover, phage HEf13 showed an effective lytic activity against E. faecalis on human dentin ex vivo infection model. Whole genome analysis demonstrated that the phage HEf13 genome contains 57,811 bp of double-stranded DNA with a GC content of 40.1% and 95 predicted open reading frames (ORFs). Annotated functional ORFs were mainly classified into four groups: DNA replication/packaging/regulation, phage structure, host cell lysis, and additional functions such as RNA transcription. Comparative genomic analysis demonstrated that phage HEf13 is a novel phage that belongs to the Sap6virus lineage. Furthermore, the results of multiple sequence alignment showed that polymorphism of phage infection protein of E. faecalis (PIPEF) contributes to determine the host specificity of phage HEf13 against various E. faecalis strains. Collectively, these results suggest that phage HEf13 has characteristics of a lytic phage, and is a potential therapeutic agent for treatment or prevention of E. faecalis-associated infectious diseases.

Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli FORC_035 Reveals the Essential Role of Iron Acquisition for Survival in Canola Sprouts and Water Dropwort.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that poses a serious threat to humans. Although EHEC is problematic mainly in food products containing meat, recent studies have revealed that many EHEC-associated foodborne outbreaks were attributable to spoiled produce such as sprouts and green leafy vegetables. To understand how EHEC adapts to the environment in fresh produce, we exposed the EHEC isolate FORC_035 to canola spouts (Brassica napus) and water dropwort (Oenanthe javanica) and profiled the transcriptome of this pathogen at 1 and 3 h after incubation with the plant materials. Transcriptome analysis revealed that the expression of genes associated with iron uptake were down-regulated during adaptation to plant tissues. A mutant strain lacking entB, presumably defective in enterobactin biosynthesis, had growth defects in co-culture with water dropwort, and the defective phenotype was complemented by the addition of ferric ion. Furthermore, gallium treatment to block iron uptake inhibited bacterial growth on water dropwort and also hampered biofilm formation. Taken together, these results indicate that iron uptake is essential for the fitness of EHEC in plants and that gallium can be used to prevent the growth of this pathogen in fresh produce.

Analysis of Microbiota in Bellflower Root, Platycodon grandiflorum, Obtained from South Korea.

Bellflower root (Platycodon grandiflorum), which belongs to the Campanulaceae family, is a perennial grass that grows naturally in Korea, northeastern China, and Japan. Bellflower is widely consumed as both food and medicine owing to its high nutritional value and potential therapeutic effects. Since foodborne disease outbreaks often come from vegetables, understanding the public health risk of microorganisms on fresh vegetables is pivotal to predict and prevent foodborne disease outbreaks. We investigated the microbial communities on the bellflower root (n = 10). 16S rRNA gene amplicon sequencing targeting the V6-V9 regions of 16S rRNA genes was conducted via the 454-Titanium platform. The sequence quality was checked and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using the weighted Fast UniFrac distance. The average number of sequence reads generated per sample was 67,192 sequences. At the phylum level, bacterial communities from the bellflower root were composed primarily of Proteobacteria, Firmicutes, and Actinobacteria in March and September samples. Genera Serratia, Pseudomonas, and Pantoea comprised more than 54% of the total bellflower root bacteria. Principal coordinate analysis plots demonstrated that the microbial community of bellflower root in March samples was different from those in September samples. Potential pathogenic genera, such as Pantoea, were detected in bellflower root samples. Even though further studies will be required to determine if these species are associated with foodborne illness, our results indicate that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria on fresh vegetables.

Characterization of LysPBC4, a novel Bacillus cereus-specific endolysin of bacteriophage PBC4.

Bacillus cereus is a spore-forming, Gram-positive bacterium and is a major food-borne pathogen. A B. cereus-specific bacteriophage PBC4 was isolated from the soil of a stock farm, and its genome was analyzed. PBC4 belongs to the Siphoviridae family and has a genome consisting of 80 647-bp-long double-stranded DNA, including 123 genes and two tRNAs. LysPBC4, the endolysin of PBC4, has an enzymatically active domain (EAD) on its N-terminal region and a putative cell wall-binding domain (CBD) on its C-terminal region, respectively. Although the phage PBC4 showed a very limited host range, LysPBC4 could lyse all of the B. cereus strains tested. However, LysPBC4 did not kill other bacteria such as B. subtilis or Listeria, indicating that the endolysin has specific lytic activity against the B. cereus group species. Furthermore, LysPBC4_CBD fused with enhanced green fluorescent protein (EGFP) could decorate limited strains of B. cereus group, suggesting that the LysPBC4_CBD may be a promising material for specific detection of B. cereus.

Complete genome sequence of Staphylococcus equorum KS1039 isolated from Saeu-jeotgal, Korean high-salt-fermented seafood.

Staphylococcus equorum KS1039 was isolated from a form of traditional Korean high-salt-fermented seafood called Saeu-jeotgal, and exhibited growth at a NaCl (w/v) concentration of 25%. Comparative genome analyses with two other strains revealed the presence of two potassium voltage-gated channel genes uniquely in KS1039, which might be involved in salt tolerance. This first complete genome sequence of the species will increase our understanding of the genetic factors allowing it to be safely consumed by humans and to inhabit high-salt environments.