Characterization of Salmonella species from poultry slaughterhouses in South Korea: carry-over transmission of Salmonella Thompson ST292 in slaughtering process.

IMPORTANCE: Salmonella outbreaks linked to poultry meat have been reported continuously worldwide. Therefore, Salmonella contamination of poultry meats in slaughterhouses is one of the critical control points for reducing disease outbreaks in humans. OBJECTIVE: This study examined the carry-over contamination of Salmonella species through the entire slaughtering process in South Korea. METHODS: From 2018 to 2019, 1,097 samples were collected from the nine slaughterhouses distributed nationwide. One hundred and seventeen isolates of Salmonella species were identified using the invA gene-specific polymerase chain reaction, as described previously. The serotype, phylogeny, and antimicrobial resistance of isolates were examined. RESULTS: Among the 117 isolates, 93 were serotyped into Salmonella Mbandaka (n = 36 isolates, 30.8%), Salmonella Thompson (n = 33, 28.2%), and Salmonella Infantis (n = 24, 20.5%). Interestingly, allelic profiling showed that all S. Mbandaka isolates belonged to the lineage of the sequence type (ST) 413, whereas all S. Thompson isolates were ST292. Moreover, almost all S. Thompson isolates (97.0%, 32/33 isolates) belonging to ST292 were multidrug-resistant and possessed the major virulence genes whose products are required for full virulence. Both serotypes were distributed widely throughout the slaughtering process. Pulsed-field gel electrophoretic analysis demonstrated that seven S. Infantis showed 100% identities in their phylogenetic relatedness, indicating that they were sequentially transmitted along the slaughtering processes. CONCLUSIONS AND RELEVANCE: This study provides more evidence of the carry-over transmission of Salmonella species during the slaughtering processes. ST292 S. Thompson is a potential pathogenic clone of Salmonella species possibly associated with foodborne outbreaks in South Korea.

Combined antimicrobial effect of two peptide nucleic acids against Staphylococcus aureus and S. pseudintermedius veterinary isolates.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the major etiological agents of staphylococcal infections in humans, livestock, and companion animals. The misuse of antimicrobial drugs has led to the emergence of antimicrobial-resistant Staphylococcus spp., including methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP). One novel therapeutic approach against MRSA and MRSP is a peptide nucleic acid (PNA) that can bind to the target nucleotide strands and block expression. Previously, two PNAs conjugated with cell-penetrating peptides (P-PNAs), antisense PNA (ASP)-cmk and ASP-deoD, targeting two essential genes in S. aureus, were constructed, and their antibacterial activities were analyzed. OBJECTIVES: This study analyzed the combined antibacterial effects of P-PNAs on S. aureus and S. pseudintermedius clinical isolates. METHODS: S. aureus ATCC 29740 cells were treated simultaneously with serially diluted ASP-cmk and ASP-deoD, and the minimal inhibitory concentrations (MICs) were measured. The combined P-PNA mixture was then treated with S. aureus and S. pseudintermedius veterinary isolates at the determined MIC, and the antibacterial effect was examined. RESULTS: The combined treatment of two P-PNAs showed higher antibacterial activity than the individual treatments. The MICs of two individual P-PNAs were 20 and 25 µM, whereas that of the combined treatment was 10 µM. The application of a combined treatment to clinical Staphylococcus spp. revealed S. aureus isolates to be resistant to P-PNAs and S. pseudintermedius isolates to be susceptible. CONCLUSIONS: These observations highlight the complexity of designing ASPs with high efficacy for potential applications in treating staphylococcal infections in humans and animals.

Acid tolerance of enterohemorrhagic Escherichia coli O157:H7 strain ATCC 43894 and its relationship with a large virulence plasmid pO157.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes a severe intestinal infection including hemolytic uremic syndrome in humans. Various factors contribute to its pathogenesis, including a large virulence plasmid pO157. This F-like 92-kb plasmid is isolated in virtually all clinical EHEC isolates, and is considered a hallmark of EHEC virulence. A previous report stated that removal of pO157 from EHEC ATCC 43894 induced overexpression of GadAB that are essential in glutamate-dependent acid resistance (GDAR) system, yet the mechanism remains elusive. Based on this observation, we surmised that pO157 is involved in the regulation of GDAR system. We comparatively analyzed 43894 and its pO157-cured (ΔpO157) mutant 277 for i) their acid resistance, ii) changes in the transcriptional profiles and iii) expression of GDAR associated genes/proteins. Survivability of 43894 upon exposure to acidic conditions was significantly lower than the ΔpO157 mutant. In addition, RNA-sequencing revealed that genes involved in GDAR were significantly down-regulated in 43894 when compared to the ΔpO157 mutant. Exogenous expression of GadE in 43894 led to expression of GadAB, suggesting possible intervention of pO157 in GDAR regulation. Despite these findings, reintroduction of pO157 into 277 did not reverted Gad overexpression. Likewise, removing pO157 from 43894 using the plasmid incompatibility method did not induce Gad overexpression as shown in 277. Taken together, the results suggest that variation in acid resistance among EHEC isolates exists, and the large virulence plasmid pO157 has no effect on weak acid resistance phenotype displayed in 43894.

Development of antisense peptide-peptide nucleic acids against fluoroquinolone-resistant Escherichia coli.

BACKGROUND: Fluoroquinolones (FQs) are potent and broad-spectrum antibiotics commonly used to treat MDR bacterial infections, but bacterial resistance to FQs has emerged and spread rapidly around the world. The mechanisms for FQ resistance have been revealed, including one or more mutations in FQ target genes such as DNA gyrase (gyrA) and topoisomerase IV (parC). Because therapeutic treatments for FQ-resistant bacterial infections are limited, it is necessary to develop novel antibiotic alternatives to minimize or inhibit FQ-resistant bacteria. OBJECTIVES: To examine the bactericidal effect of antisense peptide-peptide nucleic acids (P-PNAs) that can block the expression of DNA gyrase or topoisomerase IV in FQ-resistant Escherichia coli (FRE). METHODS: A set of antisense P-PNA conjugates with a bacterial penetration peptide were designed to inhibit the expression of gyrA and parC and were evaluated for their antibacterial activities. RESULTS: Antisense P-PNAs, ASP-gyrA1 and ASP-parC1, targeting the translational initiation sites of their respective target genes significantly inhibited the growth of the FRE isolates. In addition, ASP-gyrA3 and ASP-parC2, which bind to the FRE-specific coding sequence within the gyrA and parC structural genes, respectively, showed selective bactericidal effects against FRE isolates. CONCLUSIONS: Our results demonstrate the potential of targeted antisense P-PNAs as antibiotic alternatives against FQ-resistance bacteria.

Recent Advancements in Technologies to Detect Enterohaemorrhagic Escherichia coli Shiga Toxins.

Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.

Mutating both relA and spoT of enteropathogenic Escherichia coli E2348/69 attenuates its virulence and induces interleukin 6 in vivo.

Here, we report for the first time that disrupting both relA and spoT genes in enteropathogenic Escherichia coli E2348/69 can attenuate its virulence and significantly induce interleukin 6 (IL-6) in vivo. Our experimental analyses demonstrated that an E2348/69 ΔrelAΔspoT double mutant strain derepressed the expression of type IV bundle forming pilus (BFP) and repressed the expression of glutamate decarboxylase (GAD) and locus of enterocyte effacement (LEE). Whole genome-scale transcriptomic analysis revealed that 1,564 EPEC genes were differentially expressed in the ΔrelAΔspoT double mutant strain (cut-off > two-fold). Such depletion of relA and spoT attenuated the virulence of E2348/69 in a Caenorhabditis elegans infection model. Surprisingly, IL-6 was highly induced in porcine macrophages infected with the ΔrelAΔspoT double mutant strain compared to those with its wildtype strain. Coinciding with these in vitro results, in vivo murine peritoneal challenge assays showed high increase of IL-6 and improved bacterial clearance in response to infection by the ΔrelAΔspoT double mutant strain. Taken together, our data suggest that relA and spoT play an essential role in regulating biological processes during EPEC pathogenesis and that their depletion can affect host immune responses by inducing IL-6.

Pseudomonas stutzeri PM101005 inhaled with atmospheric particulate matter induces lung damage through inflammatory responses.

Atmospheric particulate matter (PM) contains a mixture of chemical and biological elements that pose threat to human health by increasing susceptibility to respiratory diseases. Although the identification of the microorganisms composing the PM has been assessed, their immunological impacts are still questionable. Here, we examined the mechanisms responsible for the pathogenicity of Pseudomonas stutzeri PM101005 (PMPS), a bacterium isolated from fine dust, in lung epithelial cells, alveolar cells, and macrophages. Relative to its comparative strain Pseudomonas stutzeri (PS), infections with PMPS induced higher production of inflammatory cytokines and chemokines, mediated by the activation of NF-κB and MAPK signaling pathways. Additionally, with three-dimensional (3D) airway spheroids which mimic the human bronchial epithelium, we confirmed that PMPS infections lead to relatively higher induction of pro-inflammatory cytokines than PM infections. Consistent results were observed in murine models as the infections with PMPS provoked greater inflammatory responses than the infections with PS. These PMPS-induced responses were mediated by the signaling pathways of the Toll-like receptors (TLRs), which regulated PMPS infection and played an important role in the expression of the antibiotic peptide ß-defensin 3 (BD3) that suppressed PMPS proliferation. Moreover, PM pretreatment enhanced inflammatory responses and tissue damage of PMPS, while reducing BD3 expression. Overall, these results indicate that PM-isolated PMPS induce TLR-mediated inflammatory responses in lung tissues, and contributes to the understanding of the etiology of PM-induced respiratory damage.

Characterization of transcriptional activities at a divergent promoter of the type VI secretion system in enterohemorrhagic Escherichia coli O157:H7.

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 ß-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.

Pathophysiology of enteropathogenic Escherichia coli during a host infection.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. However, sporadic outbreaks caused by this microorganism in developed countries are frequently reported recently. As an important zoonotic pathogen, EPEC is being monitored annually in several countries. Hallmark of EPEC infection is formation of attaching and effacing (A/E) lesions on the small intestine. To establish A/E lesions during a gastrointestinal tract (GIT) infeciton, EPEC must thrive in diverse GIT environments. A variety of stress responses by EPEC have been reported. These responses play significant roles in helping E. coli pass through GIT environments and establishing E. coli infection. Stringent response is one of those responses. It is mediated by guanosine tetraphosphate. Interestingly, previous studies have demonstrated that stringent response is a universal virulence regulatory mechanism present in many bacterial pathogens including EPEC. However, biological signficance of a bacterial stringent response in both EPEC and its interaction with the host during a GIT infection is unclear. It needs to be elucidated to broaden our insight to EPEC pathogenesis. In this review, diverse responses, including stringent response, of EPEC during a GIT infection are discussed to provide a new insight into EPEC pathophysiology in the GIT.

A Novel Peptide Nucleic Acid against the Cytidine Monophosphate Kinase of S. aureus Inhibits Staphylococcal Infection In Vivo.

Here, we report a novel bactericidal peptide nucleic acid (PNA) that can induce the antisense effect on the cytidine monophosphate kinase (Cmk) of Staphylococcus aureus, a putative essential component for bacterial species. Based on the genome sequence of S. aureus N315, a set of PNA conjugates with a bacterial penetration peptide, (KFF)3K, were synthesized to target the seven potentially essential genes (cmk, deoD, ligA, smpB, glmU, pyrH, and ftsA) and further evaluated for their antibacterial properties in vitro as well as in vivo. The results demonstrated that two peptide-conjugated PNAs (P-PNAs), antisense P-PNA (ASP)-cmk1 and ASP-deoD1, targeting either the cmk or the deoD genes, had the strongest inhibitory effects on the growth of S. aureus ATCC 29740 (a bovine mastitic milk isolate) in a dose-dependent manner. In vivo application of ASP-cmk1 resulted in a significant reduction of bacterial loads in mice intraperitoneally infected with a sublethal dose of S. aureus. Moreover, ASP-cmk1 significantly increased the survival rate of the breast-fed infant mice after intramammary infection of the lactating CD-1 mice. Taken together, our characterization of ASP-cmk1 demonstrated its bactericidal activity against S. aureus as well as its effectiveness in vivo.

Molecular epidemiology of sequence type 33 of Shiga toxin-producing Escherichia coli O91:H14 isolates from human patients and retail meats in Korea.

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.

Physical properties and biological/odontogenic effects of an experimentally developed fast-setting α-tricalcium phosphate-based pulp capping material.

BACKGROUND: Recently, fast-setting α-tricalcium-phosphate (TCP) cement was developed for use in the pulp capping process. The aim of this study was to investigate the physical properties and biological effects of α-TCP cement in comparison with mineral trioxide aggregate (MTA). METHODS: We measured the setting time, pH values, compressive strength, and solubility of the two materials. We evaluated biocompatibility on the basis of cell morphology and a viability test using human dental pulp cells (hDPCs). Chemical composition of each material was analyzed by energy dispersive x-ray spectroscopic (EDS) analysis. The expression of odontogenic-related genes was evaluated by Western blotting and immunofluorescence. The calcified nodule formation was measured by Alizarin red staining. We performed the pulp capping procedure on rat teeth for histological investigation. The data were analyzed by an independent t-test for physical properties, one-way ANOVA for biological effects, and the Mann-Whitney U test for tertiary dentin formation. A P value of less than 0.05 was considered statistically significant for all tests. RESULTS: The setting time, pH values, and compressive strength of α-TCP was lower than that of MTA (P < 0.05); however, the solubility of α-TCP was higher than that of MTA (P < 0.05). The resultant cell viability observed with the two materials was similar (P > 0.05). Scanning electron microscopy (SEM) revealed that cells attached to both materials were flat and had cytoplasmic extensions. The expression of odontogenic-related markers and mineralized nodule formation were higher in the two experimental groups compared to the control group (P < 0.05). Continuous tertiary dentin was formed underneath the capping materials in all samples of the tested groups. CONCLUSIONS: Our study demonstrated that the α-TCP exhibited biocompatibility and odontogenicity comparable to MTA, whereas it had a quicker setting time.