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The type VI secretion system (T6SS) is a novel secretion system found in many Gram-negative bacteria that plays a role in bacterial competition, virulence, and host immune evasion. The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 has a single functional T6SS gene cluster. In this study, we attempted to characterize the transcriptional pattern of the T6SS effector gene Z0264 in EDL933. Transcriptional analyses showed that Z0264 and other T6SS genes were transcribed in vitro in a growth-phase-dependent manner, but Z0264 was not secreted in the rich medium. Using adapter- and radioactivity-free transcription start site analysis, we identified an overlapping divergent promoter between Z0264 and Z0265. A β-galactosidase assay with truncated promoter regions showed that the divergent promoter is functional. In addition, we demonstrated the role of H-NS as a repressor in the transcription of Z0264. Notably, the cDNA PCR assay showed that the mRNA transcript from the Z0264 promoter did not include the entire main T6SS cluster, suggesting segmented gene expression by multiple promoters in the T6SS cluster. In conclusion, we identified a divergent promoter for Z0264 located in the T6SS cluster of EDL933 and characterized its in vitro transcriptional activity during growth. Our findings provide insights and a preliminary understanding of the regulatory mechanisms underlying T6SS transcription.

Salmonella is one of the most important bacterial pathogens responsible for many zoonotic food-related infectious diseases. Quantitative detection of the foodborne Salmonella contamination in various food sources is therefore critical for preventing the related disease outbreaks. In this study, we developed and evaluated a reliable real-time polymerase chain reaction (RT-PCR) assay to detect the Salmonella contamination quantitatively. The experimental results showed that our invA gene-specific quantitative RT-PCR (qRT-PCR) assay provides a strong correlation between the Cq values and the direct plate counts of Salmonella species in the artificially formulated samples. Further study may be necessary to identify more accurate correlation and equation that can apply to Salmonella spp.

Antibiotics have long been used for anti-infective control of bacterial infections, growth promotion in husbandry, and prophylactic protection against plant pathogens. However, their inappropriate use results in the emergence and spread of multiple drug resistance (MDR) especially among various bacterial populations, which limits further administration of conventional antibiotics. Therefore, the demand for novel anti-infective approaches against MDR diseases becomes increasing in recent years. The peptide nucleic acid (PNA)-based technology has been proposed as one of novel anti-infective and/or therapeutic strategies. By definition, PNA is an artificially synthesized nucleic acid mimic structurally similar to DNA or RNA in nature and linked one another via an unnatural pseudo-peptide backbone, rendering to its stability in diverse host conditions. It can bind DNA or RNA strands complimentarily with high affinity and sequence specificity, which induces the target-specific gene silencing by inhibiting transcription and/or translation. Based on these unique properties, PNA has been widely applied for molecular diagnosis as well as considered as a potential anti-infective agent. In this review, we discuss the general features of PNAs and their application to various bacterial pathogens as new anti-infective or antimicrobial agents.

Enteropathogenic

This study determined the serotyping and genotyping properties of Escherichia coli strains isolated from kimchi and various raw vegetables used for kimchi preparation. In addition, the kinetic behavior of E. coli strains in kimchi during fermentation was also determined using a predictive microbiological model. The study results revealed that E. coli isolated from napa cabbage (3.3%; 1/30) was enterohemorrhagic E. coli (O6:H34), and eight typical colonies isolated from kimchi (15%; 6/40) were enteropathogenic E. coli (H8, H8, H12, H34, H30, O20:H39, H39, and H12). The genetic correlation of the strains did not show close genetic correlations. On the other hand, the kinetic behavior of E. coli strains in kimchi during fermentation using a predictive Baranyi model (primary model) and a polynomial equation (secondary model), followed by validation by calculating root mean square error (RMSE), revealed that the pathogenic E. coli cell counts increased (with RMSE of 0.280 in growth curve) in the early stage of fermentation and decreased (with RMSE of 0.920 in death curve) thereafter depending on fermentation temperature. Therefore, this finding indicated that pathogenic E. coli isolated from kimchi and related vegetables underwent proliferation at the beginning of fermentation, which decreased thereafter. Thus, these results of this study suggest intake of sufficiently fermented kimchi to prevent potential foodborne illness due to pathogenic E. coli.

Turmeric is known for its ability to enhance immunity via anti-inflammatory and anti-oxidant effects. Salmonella enterica species contain a large number of pathogenic serotypes that are adapted to a broad range of vertebrate hosts. Our previous study revealed that bioprocessed polysaccharides from the liquid culture Lentinus edodes fungal mycelia containing turmeric (BPP-turmeric) is able to alter chicken macrophage responses and increases chick survival against Salmonella enterica infection. In this study, we examined the immunomodulatory effects of BPP-turmeric on the porcine macrophage 3D4/31 cell line infected with Salmonella enterica subsp. enterica serovar Choleraesuis (S. Choleraesuis) or S. Enteritidis. Our experimental analyses demonstrated that BPP-turmeric (i) does not alter phagocytic and killing activity of 3D4/31 against either Salmonella serotypes, but that it (ii) represses mRNA transcription of interleukin (IL)-6, IL-8, and tumor necrosis factor α in response to Salmonella infection. Collectively, these results imply that BPP-turmeric has an immunomodulatory effect that represses pro-inflammatory cytokine expression in porcine macrophages, suggesting that it may protect swine from salmonellosis via controlling Salmonella-induced hyperinflammation.

Global dissemination of non-typhoidal Salmonella producing extended-spectrum β-lactamase (ESBL) is a public-health concern. Recently, the prevalence of Salmonella spp. resistant to third-generation cephalosporins has been increasing in food-producing animals in Korea. In this study, we investigated resistance mechanisms and molecular characteristics of S. Virchow isolates resistant to extended-spectrum cephalosporins (ESCs). We obtained 265 S. Virchow isolates from fecal and carcasses samples of cattle (n = 2), pigs (n = 7), and chickens (n = 256) during 2010-2017, and observed high ESC-resistance (63.8%, 169/265); most of the resistant isolates (96.4%) were obtained from chickens. ESC-resistant S. Virchow isolates (n = 169) showed significantly higher resistance rates to other antimicrobials (especially aminoglycosides and tetracycline, p-value0.0001), as well as prevalence of multidrug resistance, than did ESC-susceptible S. Virchow isolates (n = 96). All ESC-resistant S. Virchow produced CTX-M-15-type ESBL (n = 147) and/or CMY-2-type AmpC β-lactamase (n = 23). ESC-resistant S. Virchow represented seven pulsotypes, predominantly composed of type II (58.6%) and III (26.0%), detected in 69 farms in 10 provinces, and 33 farms in 7 provinces, respectively. Genes encoding ESC-resistance were horizontally transferred by conjugation to recipient E. coli J53; this was demonstrated in 28.8% (42/146) of bla

In this study, we investigated the efficacy of chlorine-based disinfectants (sodium hypochlorite and chlorine dioxide) against Salmonella Enteritidis planktonic cells in the presence of different level of organic substances. Also, the reduction effect against S. Enteritidis biofilms formed on stainless steel, silicone rubber, plastic, and chicken skin were evaluated. At the same effective chlorine concentration under clean condition, chlorine dioxide (ClO2) had significantly (P

Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from Salmonella Typhimurium infection through upregulated T-helper 1 immunity. Here, we report that a BPP from L. edodes mushroom mycelia liquid cultures supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against avian-adapted Salmonella Gallinarum and protects chicks against a lethal challenge from Salmonella Gallinarum. In vitro analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression or secretion profile of Salmonella Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum, and (iii) significantly activated the transcription expression of interleukin (IL)-1β, IL-10, tumor necrosis factor α, and inducible nitric oxide synthase in response to various Salmonella infections, whereas it repressed that of IL-4, IL-6, interferon-β, and interferon-γ. We also found that BPP-turmeric (0.1 g/kg of feed) as a feed additive provided significant protection to 1-day-old chicks infected with a lethal dose of Salmonella Gallinarum. Collectively, these results imply that BPP-turmeric contains biologically active component(s) that protect chicks against Salmonella Gallinarum infection, possibly by regulating macrophage immune responses. Further studies are needed to evaluate the potential efficacy of BPP-turmeric as a livestock feed additive for the preharvest control of fowl typhoid or foodborne salmonellosis.

Study design: Case report and relevant literature review. Objective: To discuss the management of severe flexion-distraction injury of the subaxial cervical spine in a multisystem trauma patient. Summary of background data: Traumatic cervical spine injury from flexion-distraction injury can cause significant instability requiring extensive instrumentation complicated by vascular and soft tissue injuries. Methods: The medical record of a patient who suffered traumatic flexion-distraction injury was reviewed for relevant clinical and radiology data. A literature review on the management of traumatic cervical injuries was performed using the PubMed database. Results: We report a case of 21-year-old woman who suffered a C5-C6 flexion-distraction injury. After she underwent anterior cervical discectomy and fusion (ACDF), her care was transferred to the senior author (S.K.) due to the severity of the distraction. The patient returned to the OR the next day and underwent removal of implants at C5 and corpectomy with anterior and posterior instrumentation. Conclusion: There are many ways to manage a flexion-distraction injury of the cervical spine. In a polytrauma patient, the surgical strategy can become complex. We present a surgical option with an acceptable outcome. Keywords: Flexion-distraction injury, Corpectomy, Expandable vertebral body cage, Subaxial cervical spine, Fusion, Traumatic cervical spine fracture, Trauma, Three-column injury, Anterior cervical instrumentation, Posterior cervical instrumentation, 360 cervical fusion

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Shiga toxins (Stxs), some of the most important virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7, are known to be induced and released by various environmental cues, such as DNA damage responses and stress-inducing chemicals. In order to investigate the possible effects of growth media on Stxs expression, we analyzed the growth kinetics and expression of Stxs (Stx1 and 2) in cells grown in Luria-Bertani (LB) and E. coli (EC) media, which are widely used for EHEC O157:H7. Through direct plating and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), it was revealed that, when EHEC O157:H7 was grown in EC medium, the total bacterial count was reduced significantly and the stx1 transcription was greatly increased during the stationary growth phase than that in LB. Here we report that bile salts and lactose, which are the two only components in EC medium that are absent in LB, function as negative and positive regulatory signals, respectively, for the transcription of both stx1 and stx2. Indeed, stx transcription was significantly increased (~5.7 and ~21.8 fold for stx1 and stx2, respectively; p < 0.05) in an EC medium lacking bile salts when compared to the normal EC. In contrast, EHEC O157:H7 grown in an EC medium lacking lactose did significantly decrease these transcriptions (~93.5 and ~4.3 fold for stx1 and stx2, respectively; p < 0.05). Consistently, stx transcription was drastically increased in an LB medium supplemented with lactose, implying that lactose might be an environmental trigger for the expression of Stxs.

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx1, stx2c, stx2e, or stx1 + stx2b) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Rapid, quantitative and sensitive detection methods are essential for prevention and risk assessment of food poisoning. In this study, we developed quantitative real-time immuno-polymerase chain reaction (qRT-iPCR), a highly sensitive and efficient method for detection of staphylococcal enterotoxin H (SEH). The qRT-iPCR is based on antigen–antibody recognition, similar to the traditional enzyme-linked immunosorbent assay (ELISA). However, unlike ELISA, the method relies on DNA probes and RT-PCR for detection rather than on a chromogenic reaction. We produced recombinant SEs and monoclonal antibodies for SEH. Then, coupled RT-PCR using DNA probes was applied. This qRT-iPCR showed a lower limit of detection (LOD) than traditional ELISA did. In the sandwich format, qRT-iPCR system could detect approximately 4.5 pg/mL of SEH. Our qRT-iPCR system simplified the detection and quantification of SEH and accelerated the process compared with traditional ELISA. To our knowledge, this is the most sensitive technique for detection of SEH reported to date. Practical Applications Sensitive and rapid commercial kits to detect and quantify staphylococcal enterotoxin H, which could not be detected by the existing kits, would be developed based on the quantitative real-time immuno-polymerase chain reaction method in this study and they can improve the process of risk assessment of food poisoning.

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.

Mesenchymal stem cell (MSC) based cell therapy has emerged as a promising therapeutic approach for treatment of several degenerative, infectious and non-infectious diseases. Numerous studies have demonstrated the remarkable immunosuppressive and antibacterial effects of MSCs both in vitro and in vivo, in animal models and in humans. However, the antibacterial effects of MSCs rely heavily on their paracrine factors rather than direct cell-to-cell contact and the effect is specific to disease and site of infection or injury. Furthermore, recent studies have demonstrated the double-edged sword effect of MSCs in bacterial infectious diseases. Despite their inherent potential for repair of damaged tissues, immunosuppression, and alleviation of various autoimmune as well as infectious diseases, MSCs also play a critical role in promoting persistent bacterial infection and disease progression. Therapeutic administration of MSCs successfully inhibited the bacterial growth and enhances survival by improved clearance of pathogenic bacteria in sepsis and pneumonic conditions. However, due to their abnormal transformation, they assist in long lasting survival and persistent infection of Mycobacterium tuberculosis (M. tuberculosis) and may also be responsible for progression of gastric cancer. This review focuses on recent advances that have broadened our understanding of MSC based therapy for bacterial diseases and provides new insight into the possible therapeutic targets of fatal bacterial diseases.

A total of 339 Enterococcus faecalis strains were isolated from pork production chain, slaughterhouses, processing plants and retails, and were compared with human infection strains for investigating the transmission from animal to human via food production chain. From slaughterhouses to retails, most of antimicrobial resistances and multidrug resistance rates decreased or were not changed significantly. Meanwhile, the prevalence rate of strong or moderate biofilm-forming isolates was highest at retails. In the result of random amplified polymorphic DNA-polymerase chain reaction analysis, there was no strain persisting along the pork production chain or being similar to human strains. Consequently, it is suggested that E. faecalis strains from pork meat would not be delivered to humans by consumption of pork. Practical Applications Through investigation of antimicrobial resistant bacteria of the pork production chain of Korea, more information and relief would be provided to consumers and its results could help policy making in public health and food safety.

Purpose: Complications from RA can involve the thoracolumbar spine and cause severe spine instability and sagittal deformity. We present a surgical strategy to restore sagittal balance in a patient with chronic rheumatoid arthritis. Methods: A retrospective chart review was performed. Results: We report the case of a 53-year-old woman with RA and severe back pain. She had dynamic instability of her lumbar spine with spondylolithesis at L3-L4 and L4-L5 as well as hypermobility at the C1-2 level without rheumatoid pannus. On 36-inch standing films, she had a sagittal vertical axis (SVA) of 219 mm. She underwent anterior and posterior instrumented fusion to restore her sagittal imbalance, which resulted in excellent relief of her back pain and marked improvement in ambulation and function. Conclusion: Patients with RA can develop severe sagittal deformity that could be the source of back pain. In patients with advanced RA, chronic inflammation could lead to ligament laxity that can result in spinal deformity under axial load. This deformity is amenable to reconstruction without the utilization of an osteotomy, which can produce excellent pain relief and restoration of function.

Aeromonas salmonicida is an important fish pathogen commonly associated with furunculosis in salmonids. Typical A. salmonicida strains have the surface virulence A-layer protein, a major virulence determinant encoded by the vapA gene. In this study, 880 chum salmon Oncorhynchus keta were collected from the east coast of Korea during 2006-2011, including 560 wild adults and 320 artificially hatched fry pools, and the presence of typical A. salmonicida was examined by PCR using the typical A. salmonicida-specific vapA gene primers. The results demonstrated that 34.5% of the samples (304/880 samples) were PCR positive, implying that a typical A. salmonicida infection is highly prevalent among chum salmon in Korea. Twenty typical A. salmonicida isolates were recovered based on their brown pigmentation on Trypticase Soy Agar (TSA) plates, which indicates the existence of the A-layer protein. Further biochemical analyses with the four randomly selected typical A. salmonicida isolates revealed some variations in their amino acid decarboxylation and carbohydrate fermentation activity. A phylogenetic analysis based on the entire vapA gene sequence suggested that the A. salmonicida isolates from chum salmon were clustered with those isolated from Atlantic salmon in Europe. Further study is needed to resolve such an interesting relationship in detail.

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Streptococcus pneumoniae, the aetiological agent of pneumonia and non-gonococcal urethritis, shares a high degree of DNA sequence identity with the viridans group of streptococci, particularlyStreptococcus mitisandStreptococcus oralis. Although their clinical and pathological manifestations are different, discrimination betweenS. pneumoniaeand its close viridans cocci relatives is still quite difficult. Suppression subtractive hybridization was performed to identify the genomic differences betweenS. pneumoniaeandS. mitis. Thirty-four resultingS. pneumoniae-specific clones were examined by sequence determination and comparative DNA sequence analysis usingblast.S. pneumoniae-specific primers were subsequently designed from one of the clonal DNA sequences containing thecpsgene (coding for capsular polysaccharide biosynthesis). The primer specificities were evaluated using 49 viridans streptococci including 26S. pneumoniae, 54 other streptococci, 14Lactococcusspecies, 14Enterococcusspecies and threeVagococcusspecies, and compared with the specificities of previously described autolysin (lytA), pneumolysin (ply), Spn9802 and Spn9828 primers. The newly designedcpsA-specific primer set was highly specific toS. pneumoniaeand was even better than the existing primers. These findings may help improve the rapid identification and differentiation ofS. pneumoniaefrom closely related members of the viridans group streptococci.

Streptococcus pneumoniae, the leading etiological agent of pneumonia, shares a high degree of DNA sequence homology with the viridans group of streptococci. The clinical and pathological manifestations may present with different features, and discrimination between S. pneumoniae and its close viridans cocci relatives, such as Streptococcus mitis and Streptococcus oralis, is still quite difficult. The 445-bp sequences of the N-terminal region of rpoA from nine S. pneumoniae, seven S. mitis, ten S. oralis, and two related strains were determined and compared with their respective 16S rRNA gene sequences to establish their usefulness in phylogenetic analysis. Pairwise comparisons of rpoA sequences among the species showed higher rates of evolution with lower similarities (92.3-100%) than those of 16S rRNA genes (96.8-100%). The rpoA-based phylogeny generated deeper branches and presented improved discriminatory resolution than the 16S rRNA gene-based phylogeny. These results show that rpoA sequences represent a consistent, but more discriminating, genomic marker than 16S rRNA gene sequences for investigating the evolutionary relationships of Streptococcus at the species level. rpoA could be a useful marker for identifying and classifying S. pneumoniae, S. mitis, and S. oralis from closely related taxa.

The purpose of this study was to investigate the relationship between the global regulatory mechanism known as quorum sensing and expression of virulence factors in Escherichia coli O157:H7. A non-polar luxS deletion was introduced into the chromosome of strain CI03J, a human clinical isolate from South Korea, to create the DeltaluxS mutant strain ML03J. Phenotypic characterization of wild-type and mutant strains demonstrated that ML03J had no obvious growth or metabolic defects on 0.2% glucose LB medium, produced a functionally-defective flagellum, and could not utilize sorbose; the biological significance of sorbose utilization is unknown. Omics-based analysis revealed the involvement of LuxS in the transcriptional activation of several flagella/chemotaxis-related genes (flhD; fliA, C, D, S, Z; cheA, Y, and Z), repression of glutamate-dependent acid resistance genes (gadAB), and expression of virulence factors including Shiga toxin, hemolysin, and SepD within the LEE pathogenicity island.