Prevalence of antibiotic-resistant Acinetobacter spp. on soil and crops collected from agricultural fields in South Korea.

The emergence of antibiotic resistance in Acinetobacter spp. is a rising public health concern worldwide. The objective of this study was to investigate the prevalence of antibiotic-resistance genes and the virulence of Acinetobacter spp. isolated from soil and crops obtained from agricultural fields in South Korea. Eight Acinetobacter spp. isolates carried various antibiotic resistance genes, such as emrAB (100%), cat/craA (100%), and aadA gene (87.5%). Minimum inhibitory concentration (MIC) analysis revealed that strains harboring antibiotic resistance genes exhibited high resistance to the respective antibiotics, such as colistin, chloramphenicol, and streptomycin. Interestingly, most of these isolates had high capability of biofilm formation and swarming motility, along with faster growth rates. Taken together, our study demonstrated that antibiotic-resistant Acinetobacter isolated from agricultural settings in South Korea not only frequently carries antibiotic resistance genes but also has virulence-related traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01496-7.

Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea.

Introduction: The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. Aim: In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. Methods: To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. Results: The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Conclusion: Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone.

Cold tolerance in Campylobacter jejuni and its impact on food safety.

Campylobacter jejuni is a major cause of foodborne illnesses worldwide and is primarily transmitted to humans through contaminated poultry meat. To control this pathogen, it is critical to understand its cold tolerance because poultry products are usually distributed in the cold chain. However, there is limited information regarding how this thermotolerant, microaerophilic pathogen can survive in cold and aerobic environments in the poultry cold chain. In this study, we investigated the cold tolerance of C. jejuni by measuring the viability of 90 C. jejuni strains isolated from retail raw chicken at 4 °C under aerobic and microaerobic conditions. Despite the microaerophilic nature of C. jejuni, under aerobic conditions, C. jejuni exhibited higher viability at 4 °C and required an extended inactivation time compared to microaerobic conditions. Some strains were highly tolerant to refrigeration temperatures and exhibited increased survival at 4 °C. These cold-tolerant strains mostly belonged to multilocus sequence typing (MLST) clonal complex (CC)-21 and CC-443, indicating that cold tolerance is associated with the phylogeny of C. jejuni. Notably, cold-tolerant strains had an increased probability of illness and were more likely to cause human infections due to their extended survival on refrigerated chicken meat compared to those sensitive to cold stress. Furthermore, the majority of cold-tolerant strains exhibited elevated aerotolerance, indicating that cold tolerance is related to aerotolerance. These findings suggest that refrigeration of chicken meat under aerobic conditions may not be effective at controlling C. jejuni and that cold-tolerant C. jejuni can pose an increased risk to food safety.

Development of sensitizer peptide-fused endolysin Lys1S-L9P acting against multidrug-resistant gram-negative bacteria.

The advent of multidrug-resistant (MDR) bacteria poses a major threat to public health, garnering attention to novel antibiotic replacements. Endolysin, a bacteriophage-derived cell wall-degrading enzyme, is a promising alternative to conventional antibiotics. However, it is challenging to control Gram-negative bacteria due to the presence of the outer membrane that shields the peptidoglycan layer from enzymatic degradation. To overcome this threshold, we constructed the fusion endolysin Lys1S-L9P by combining endolysin LysSPN1S with KL-L9P, a sensitizer peptide known to extend efficacy of antibiotics by perturbing the outer membrane of Gram-negative bacteria. In addition, we established a new endolysin purification procedure that increases solubility allowing a 4-fold increase in production yield of Lys1S-L9P. The sensitizer peptide-fused endolysin Lys1S-L9P exhibited high bactericidal effects against many MDR Gram-negative pathogens and was more effective in eradicating biofilms compared to LysSPN1S. Moreover, Lys1S-L9P showed potential for clinical use, maintaining stability at various storage temperatures without cytotoxicity against human cells. In the in vivo Galleria mellonella model, Lys1S-L9P demonstrated potent antibacterial activity against MDR Gram-negative bacteria without inducing any toxic activity. This study suggest that Lys1S-L9P could be a potential biocontrol agent to combat MDR Gram-negative bacteria.

From microbes to molecules: a review of microbial-driven antioxidant peptide generation.

Oxidative stress, arising from excess reactive oxygen species (ROS) or insufficient antioxidant defenses, can damage cellular components, such as lipids, proteins, and nucleic acids, resulting in cellular dysfunction. The relationship between oxidative stress and various health disorders has prompted investigations into potent antioxidants that counteract ROS's detrimental impacts. In this context, antioxidant peptides, composed of two to twenty amino acids, have emerged as a unique group of antioxidants and have found applications in food, nutraceuticals, and pharmaceuticals. Antioxidant peptides are sourced from natural ingredients, mainly proteins derived from foods like milk, eggs, meat, fish, and plants. These peptides can be freed from their precursor proteins through enzymatic hydrolysis, fermentation, or gastrointestinal digestion. Previously published studies focused on the origin and production methods of antioxidant peptides, describing their structure-activity relationship and the mechanisms of food-derived antioxidant peptides. Yet, the role of microorganisms hasn't been sufficiently explored, even though the production of antioxidant peptides frequently employs a variety of microorganisms, such as bacteria, fungi, and yeasts, which are recognized for producing specific proteases. This review aims to provide a comprehensive overview of microorganisms and their proteases participating in enzymatic hydrolysis and microbial fermentation to produce antioxidant peptides. This review also covers endogenous peptides originating from microorganisms. The information obtained from this review might guide the discovery of novel organisms adept at generating antioxidant peptides.

Characterization of thermostable bacteriophage CPD2 and its endolysin LysCPD2 as biocontrol agents against Clostridium perfringens.

Clostridium perfringens is one of the major foodborne pathogens in humans and animals. With the prevalence of antibiotic-resistant C. perfringens strains, bacteriophages and their endolysins have received considerable attention as promising alternatives to antibiotics. In this study, C. perfringens phage CPD2 was isolated from retail chicken samples. CPD2 belongs to the Podoviridae family and exhibits remarkable thermostability. While CPD2 has narrow host specificity, its endolysin LysCPD2 showed a broader lytic range, killing not only C. perfringens strains but other Gram-positive bacteria, such as B. cereus and B. subtilis. In addition, due to its exceptional thermal stability, LysCPD2 showed significant antibacterial ability against germinating C. perfringens spores during the heat activation process (75 °C for 20 min). Taken together, these results indicate that both thermostable phage CPD2 and its endolysin LysCPD2 can be used as efficient antimicrobial agents to control C. perfringens during thermal processing of foods.

Complete genome sequence of Pectobacterium brasiliense strain 21PCA_AGRO2 with antimicrobial resistance isolated from napa cabbage.

We report a complete genome of Pectobacterium brasiliense strain 21PCA_AGRO2 isolated from napa cabbage, in which the genome consists of a circular chromosome comprising 4,919,671 bp with 4,399 coding DNA sequences, 22 rRNA genes, 77 tRNA genes, and 9 noncoding RNA genes.

Characterization and genomic study of EJP2, a novel jumbo phage targeting antimicrobial resistant Escherichia coli.

The emergence of antimicrobial resistance (AMR) Escherichia coli has noticeably increased in recent years worldwide and causes serious public health concerns. As alternatives to antibiotics, bacteriophages are regarded as promising antimicrobial agents. In this study, we isolated and characterized a novel jumbo phage EJP2 that specifically targets AMR E. coli strains. EJP2 belonged to the Myoviridae family with an icosahedral head (120.9 ± 2.9 nm) and a non-contractile tail (111.1 ± 0.6 nm), and contained 349,185 bp double-stranded DNA genome with 540 putative ORFs, suggesting that EJP2 could be classified as jumbo phage. The functions of genes identified in EJP2 genome were mainly related to nucleotide metabolism, DNA replication, and recombination. Comparative genomic analysis revealed that EJP2 was categorized in the group of Rak2-related virus and presented low sequence similarity at the nucleotide and amino acid level compared to other E. coli jumbo phages. EJP2 had a broad host spectrum against AMR E. coli as well as pathogenic E. coli and recognized LPS as a receptor for infection. Moreover, EJP2 treatment could remove over 80% of AMR E. coli biofilms on 96-well polystyrene, and exhibit synergistic antimicrobial activity with cefotaxime against AMR E. coli. These results suggest that jumbo phage EJP2 could be used as a potential biocontrol agent to combat the AMR issue in food processing and clinical environments.

Draft Genome Sequences of Three Listeria monocytogenes Strains Isolated from Chicken Carcasses in South Korea.

While Listeria monocytogenes is a foodborne pathogen of global concern, little is known regarding L. monocytogenes populations in Asia. We report the draft genome sequences of three L. monocytogenes strains from chickens in South Korea, which could facilitate understanding the genetic diversity of the regional L. monocytogenes population.

Stimulation of Surface Polysaccharide Production under Aerobic Conditions Confers Aerotolerance in Campylobacter jejuni.

The ability of a foodborne pathogen to tolerate environmental stress critically affects food safety by increasing the risk of pathogen survival and transmission in the food supply chain. Campylobacter jejuni, a leading bacterial cause of foodborne illnesses, is an obligate microaerophile and is sensitive to atmospheric levels of oxygen. Currently, the molecular mechanisms of how C. jejuni withstands oxygen toxicity under aerobic conditions have not yet been fully elucidated. Here, we show that when exposed to aerobic conditions, C. jejuni develops a thick layer of bacterial capsules, which in turn protect C. jejuni under aerobic conditions. The presence of both capsular polysaccharides and lipooligosaccharides is required to protect C. jejuni from excess oxygen in oxygen-rich environments by alleviating oxidative stress. Under aerobic conditions, C. jejuni undergoes substantial transcriptomic changes, particularly in the genes of carbon metabolisms involved in amino acid uptake, the tricarboxylic acid (TCA) cycle, and the Embden-Meyerhof-Parnas (EMP) pathway despite the inability of C. jejuni to grow aerobically. Moreover, the stimulation of carbon metabolism by aerobiosis increases the level of glucose-6-phosphate, the EMP pathway intermediate required for the synthesis of surface polysaccharides. The disruption of the TCA cycle eliminates aerobiosis-mediated stimulation of surface polysaccharide production and markedly compromises aerotolerance in C. jejuni. These results in this study provide novel insights into how an oxygen-sensitive microaerophilic pathogen survives in oxygen-rich environments by adapting its metabolism and physiology. IMPORTANCE Oxygen-sensitive foodborne pathogens must withstand oxygen toxicity in aerobic environments during transmission to humans. C. jejuni is a major cause of gastroenteritis, accounting for 400 million to 500 million infection cases worldwide per year. As an obligate microaerophile, C. jejuni is sensitive to air-level oxygen. However, it has not been fully explained how this oxygen-sensitive zoonotic pathogen survives in aerobic environments and is transmitted to humans. Here, we show that under aerobic conditions, C. jejuni boosts its carbon metabolism to produce a thick layer of bacterial capsules, which in turn act as a protective barrier conferring aerotolerance. The new findings in this study improve our understanding of how oxygen-sensitive C. jejuni can survive in aerobic environments.

Characterization of KMSP1, a newly isolated virulent bacteriophage infecting Staphylococcus aureus, and its application to dairy products.

Staphylococcus aureus is one of the major pathogens causing foodborne outbreaks and severe infections worldwide. Generally, various physical and chemical treatments have been applied to control S. aureus in the food industry. However, conventional treatments usually affected food quality and often produced toxic compounds. Therefore, bacteriophage (phage), a natural antimicrobial agent, has been suggested as an alternative strategy to control foodborne pathogens including S. aureus. In this study, KMSP1, a bacteriophage infecting S. aureus was isolated from a raw milk sample and characterized. Transmission electron microscopy (TEM) analysis revealed that phage KMSP1 belongs to the Myoviridae family. Phage KMSP1 efficiently inhibited bacterial growth for >28 h post-infection. In addition, phage KMSP1 could infect a broad spectrum of S. aureus strains, including methicillin-resistant S. aureus (MRSA) strains. Whole-genome sequence analysis showed that KMSP1 is a lytic phage with the absence of genes related to lysogen formation, toxin production, and antibiotics resistance, respectively. In the genome of KMSP1, the presence of putative tail lysin containing a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain could be one of the reasons for the effective antimicrobial activity of KMSP1. Furthermore, high stability of phage KMSP1 at temperature ranging from 4 to 55 °C and pH ranging from 5 to 11, suggested its potential use in various food systems. Receptor analysis revealed that KMSP1 utilized cell wall teichoic acid (WTA), one of the major virulence factors of S. aureus, as a host receptor. Application of phage KMSP1 at an MOI of 104 achieved a significant reduction of log 8.8 CFU/mL of viable cell number in pasteurized milk and log 4.3 CFU/cm2 in sliced cheddar cheese after 24 h. Taken together, the strong antimicrobial activity of phage KMSP1 suggested that it could be developed as a biocontrol agent in dairy products to control S. aureus contamination.

Draft Genome Sequence of Listeria monocytogenes Clonal Complex 1 Strain SNU3 from South Korea.

Currently, only limited knowledge is available about the genetic diversity of the hypervirulent clonal complex 1 (CC1) of Listeria monocytogenes from Asia. In this study, we report the draft genome sequence of an L. monocytogenes CC1 strain (SNU3) from Seoul, South Korea.

Bacteriophage and endolysin engineering for biocontrol of food pathogens/pathogens in the food: recent advances and future trends.

Despite advances in modern technologies, various foodborne outbreaks have continuously threatened the food safety. The overuse of and abuse/misuse of antibiotics have escalated this threat due to the prevalence of multidrug-resistant (MDR) pathogens. Therefore, the development of new methodologies for controlling microbial contamination is extremely important to ensure the food safety. As an alternative to antibiotics, bacteriophages(phages) and derived endolysins have been proposed as novel, effective, and safe antimicrobial agents and applied for the prevention and/or eradication of bacterial contaminants even in foods and food processing facilities. In this review, we describe recent genetic and protein engineering tools for phages and endolysins. The major aim of engineering is to overcome limitations such as a narrow host range, low antimicrobial activity, and low stability of phages and endolysins. Phage engineering also aims to deter the emergence of phage resistance. In the case of endolysin engineering, enhanced antibacterial ability against Gram-negative and Gram-positive bacteria is another important goal. Here, we summarize the successful studies of phages and endolysins treatment in different types of food. Moreover, this review highlights the recent advances in engineering techniques for phages and endolysins, discusses existing challenges, and suggests technical opportunities for further development, especially in terms of antimicrobial agents in the food industry.

Characterization of the lytic phage MSP1 for the inhibition of multidrug-resistant Salmonella enterica serovars Thompson and its biofilm.

The increasing prevalence of multidrug-resistant (MDR) Salmonella is a serious public health threat. Intervention strategies available to control Salmonella mostly target Salmonella enterica serovars Typhimurium and Enteritidis, and little has been investigated to control serovars in serogroup C, such as S. enterica serovar Thompson, despite their increasing prevalence. Here, we isolated phages targeting MDR S. Thompson and characterized the antimicrobial activities of MSP1 phage, a virulent phage with a broad host range. MSP1 phage strongly infected S. Thompson and S. Mbandaka isolates from retail chicken and also other serovars, including Dublin, Enteritidis, Heidelberg, Paratyphi, and Typhimurium. MSP1 phage was able to inhibit the biofilm formation on stainless steel and glass formation by around 42.7-47.9 %. MSP1 phage was robust to withstand wide ranges of pH (4-12) and temperature (30-60 °C), and no genes associated with antibiotic resistance and virulence were found in the phage genome, suggesting that this phage is suitable for food application. When MSP1 phage was tested on foods (chicken meat and milk), MSP1 phage significantly reduced the level of MDR S. Thompson below the detection limit. Our findings suggest that MSP1 phage is a promising antimicrobial agent for the control of food contamination by MDR S. Thompson.

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.

Sugar Modification of Wall Teichoic Acids Determines Serotype-Dependent Strong Biofilm Production in Listeria monocytogenes.

Biofilm production is responsible for persistent food contamination by Listeria monocytogenes, threatening food safety and public health. Human infection and food contamination with L. monocytogenes are caused primarily by serotypes 1/2a, 1/2b, and 4b. However, the association of biofilm production with phylogenic lineage and serotype has not yet been fully understood. In this study, we measured the levels of biofilm production in 98 clinical strains of L. monocytogenes at 37°C, 25°C, and 4°C. The phylogenetic clusters grouped by core genome multilocus sequence typing (cgMLST) exhibited association between biofilm production and phylogenetic lineage and serotype. Whereas clusters 1 and 3 consisting of serotype 4b strains exhibited weak biofilm production, clusters 2 (serotype 1/2b) and 4 (serotype 1/2a) were composed of strong biofilm formers. Particularly, cluster 2 (serotype 1/2b) strains exhibited the highest levels of biofilm production at 37°C, and the levels of biofilm production of cluster 4 (serotype 1/2a) strains were significantly elevated at all tested temperatures. Pan-genome analysis identified 22 genes unique to strong biofilm producers, most of which are related to the synthesis and modification of teichoic acids. Notably, a knockout mutation of the rml genes related to the modification of wall teichoic acids with l-rhamnose, which is specific to serogroup 1/2, significantly reduced the level of biofilm production by preventing biofilm maturation. Here, the results of our study show that biofilm production in L. monocytogenes is related to phylogeny and serotype and that the modification of wall teichoic acids with l-rhamnose is responsible for serotype-specific strong biofilm formation in L. monocytogenes. IMPORTANCE Biofilm formation on the surface of foods or food-processing facilities by L. monocytogenes is a serious food safety concern. Here, our data demonstrate that the level of biofilm production differs among serotypes 1/2a, 1/2b, and 4b depending on the temperature. Furthermore, sugar decoration of bacterial cell walls with l-rhamnose is responsible for strong biofilm production in serotypes 1/2a and 1/2b, commonly isolated from foods and listeriosis cases. The findings in this study improve our understanding of the association of biofilm production with phylogenetic lineage and serotype in L. monocytogenes.

Phylogenetic Association and Genetic Factors in Cold Stress Tolerance in Campylobacter jejuni.

Campylobacter jejuni is a major foodborne pathogen transmitted to humans primarily via contaminated poultry meat. Since poultry meat is generally processed, distributed, and stored in the cold chain, the survival of C. jejuni at refrigeration temperatures crucially affects human exposure to C. jejuni. Here, we investigated genetic factors associated with cold stress tolerance in C. jejuni. Seventy-nine C. jejuni strains isolated from retail raw chicken exhibited different survival levels at 4°C for 21 days. Multilocus sequence typing (MLST) clonal complex 21 (CC-21) and CC-443 were dominant among cold stress-tolerant strains, whereas CC-45 was common among cold stress-sensitive strains. Genome-wide average nucleotide identity (ANI) analysis identified a phylogenetic cluster associated with cold stress tolerance. Moreover, a pangenome analysis revealed 58 genes distinctively present in the cold stress-tolerant phylogenetic cluster. Among these 58 genes, cfrA, encoding the ferric enterobactin receptor involved in ion transport and metabolism, was selected for further analysis. Remarkably, the viability of a ΔcfrA mutant at 4°C was significantly decreased, while the levels of total reactive oxygen species and intracellular iron exceeded those of the wild type. Additionally, a knockout mutation of cfrA also significantly decreased the viability of three cold stress-tolerant isolates at 4°C, confirming the role of cfrA in cold stress tolerance. The results of this study demonstrate that unique phylogenetic clusters of C. jejuni associated with cold stress tolerance exist and that cfrA is a genetic factor contributing to cold stress tolerance in C. jejuni. IMPORTANCE The tolerance of foodborne pathogens to environmental stresses significantly affects food safety. Several studies have demonstrated that C. jejuni survives extended exposures to low temperatures, but the mechanisms of cold stress tolerance are not fully understood. Here, we demonstrate that C. jejuni strains in certain phylogenetic groups exhibit increased tolerance to cold stress. Notably, cfrA is present in the phylogenetic cluster associated with cold stress tolerance and plays a role in the survival of C. jejuni at low temperatures by alleviating oxidative stress. This is the first study to discover phylogenetic associations involving cold stress tolerance and to identify genetic elements conferring cold stress tolerance to C. jejuni.

Isocitrate binds to the itaconic acid-responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis.

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella. The RipR regulatory domain structure exhibited the typical dimeric arrangement with the putative ligand-binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand-binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence.

Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P < 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence. IMPORTANCE Pectobacterium carotovorum subsp. carotovorum is a phytopathogen that causes soft rot disease in various crops by producing plant cell wall-degrading enzymes (PCWDEs). Although antibiotics and copper-based pesticides have been extensively applied to inhibit P. carotovorum subsp. carotovorum, the emergence of antibiotic-resistant bacteria and demand for harmless antimicrobial products have emphasized the necessity of finding alternative therapeutic strategies. To address this problem, we developed a phage cocktail consisting of three P. carotovorum subsp. carotovorum-specific phages that recognize colanic acids and flagella of P. carotovorum subsp. carotovorum. The phage cocktail treatments significantly decreased P. carotovorum subsp. carotovorum populations, as well as soft rot symptoms in napa cabbage. Simultaneously, they resulted in virulence attenuation in phage-resistant P. carotovorum subsp. carotovorum, which was represented by decreased PCWDE production and decreased flagellum-mediated swimming motility. These results suggested that preparations of phage cocktails targeting multiple receptors would be an effective approach to biocontrol of P. carotovorum subsp. carotovorum in crops.