Seroepidemiologic evidence of Q fever and associated factors among workers in veterinary service laboratory in South Korea.

The incidence of Q fever has rapidly increased in South Korea since 2015. This study was undertaken to investigate the seroprevalence and seroreactivity of Q fever and the risk factors associated with its seroprevalence among workers in the veterinary service laboratory (VSL) in South Korea. This seroepidemiologic study was conducted in a total of 661 human subjects out of 1,328 subjects working in 50 VSL existing in South Korea between July 15 and July 29, 2019. Data were collected by administering survey questionnaires and by analyzing collected blood samples to determine the presence of antibodies against Coxiella burnetii. The seroprevalence and seroreactivity of C. burnetii infection were determined based on serum titers as (phase II IgG ≥1:256 and/or IgM ≥1:16) and (phase II IgG ≥1:16 and/or IgM ≥1:16) as determined by indirect immunofluorescent assay. Work, work environment, behavioral risk and protective factors associated with seroprevalence of Q fever were assessed by employing multivariable logistic regression analysis. Among the 661, the seroprevalence and seroreactivity of C. burnetii infection were 7.9% and 16.0%, respectively. Multivariate logistic regression analysis showed the risk factors significantly associated with seroprevalence were the antemortem inspection of cattle, goats, or sheep (APR (adjusted prevalence ratio), 2.52; 95% CI, 1.23-4.70)), animal blood splashed into or around eyes (APR, 2.24; 95% CI, 1.04-4.41), and contact with animals having Q fever (APR, 6.58; 95% CI, 3.39-10.85) during the previous year. This study suggests the need for precautions when contact with cattle, goats, or sheep is expected, especially during the antemortem inspection, when dealing with C. burnetii infected animals, or when there is a risk of ocular contact with animal derivatives. Therefore, we recommend the consistent use of appropriate personal protective equipment and other protective measures including PPE treatment and washing of body surfaces after work to prevent C. burnetii infections among VSL staff in South Korea.

The prevalence and characteristics of Shiga toxin-producing Escherichia coli isolated by the enteric pathogens active surveillance network (Enter-Net) in the Republic of Korea, 2009-2018.

OBJECTIVE: Shiga toxin-producing Escherichia coli (STEC) is a water- and food-borne pathogenic agent that causes diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and end-stage renal disease. As the annual incidence of STEC increases, disease control is also becoming important in Korea. In this study, we aimed to analyze the incidence trends and characteristics of STEC isolated from diarrheal patients over 10 years. METHODS: From 2009 to 2018, STECs were collected by the Enteric Pathogens Active Surveillance Network (Enter-Net) and analyzed according to clinical epidemiological information (month of isolation, age, and sex of patient), O serogroup, and shiga toxin type. Shiga toxin genes (stx1 and stx2) and O serogroups of isolates were determined using multiplex PCR and an agglutination method with the available O antisera, respectively. RESULTS: A total of 418 strains were isolated over 10 years. The isolation rate according to age group and season was highest in children ≤4 years old (38.1%) and in the summer season (June to August). Among the 418 isolates, the major serogroups were divided O157 (20.3%), O103 (13.6%), O26 (7.7%), O111 (5.5%), O91 (4.3%), O108 (2.4%), and O8 (2.2%). The most frequently isolated O157 showed a lower isolation rate compared to that isolated from other developed countries. The profiles of stx genes were distinct among serogroups. In O157 and O91, stx1+stx2 was detected more frequently than either stx1 or stx2 alone. Particularly, most of the O157 (98%) isolates harbored the stx2 gene, which is an important factor in severe diseases, including HUS. In O103, O26, O111, and O108, stx1-only was more frequently present than stx2-only or stx1+stx2. CONCLUSIONS: As a result of analyzing domestic STECs collected through Enter-Net, it was confirmed that patients ≤4 years of age and in the summer months require attention, and that STEC with a serogroup of O157 is highly likely to cause diseases such as HUS. Therefore, the pathogen active surveillance network for characterization and provision of STEC isolates must be operated continuously.

Emerging high-level ciprofloxacin-resistant Salmonella enterica serovar typhi haplotype H58 in travelers returning to the Republic of Korea from India.

In Korea, typhoid fever is a rare disease due to improved living standards. However, typhoid fever remains a major burden in developing countries and regions, such as India and Southeast Asia. In this study, we isolated Salmonella Typhi (S. Typhi) from eight patients with typhoid fever who were travelers returning from India. The strains isolated were characterized by antimicrobial susceptibility profiling and whole-genome sequencing (WGS) analysis. All strains were resistant to nalidixic acid and azithromycin. Among them, four isolates were highly resistant to ciprofloxacin (MIC ≥32 µg/ml); these strains have not been confirmed in Korea PulseNet DB. According to WGS, the ciprofloxacin-resistant strains belong to the global dominant multidrug-resistant (MDR) haplotype H58 (SNP glpA C1047T, SptP protein Q185* (premature stop codon)) and do not harbor the MDR plasmid. H58-associated SNPs in membrane and metabolism genes, including yhdA, yajI, hyaE, tryE, rlpB and metH, are present. Additionally, phylogenetic analysis assigned the H58 strains to sublineage II, whereas the non-H58 strains are closely related to haplotype H50. The presence of high-level ciprofloxacin-resistant S. Typhi haplotype H58 in Korea was first confirmed as due to influx from overseas via travelers. This study provides information about intercontinental drug-resistant transmission between countries and suggests that travelers need to be careful about personal hygiene.

Construction of a bivalent vaccine against anthrax and smallpox using the attenuated vaccinia virus KVAC103.

BACKGROUND: Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. RESULTS: Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. CONCLUSIONS: We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

Co-infection With Chromosomally-Located bla CTX-M-14 and Plasmid-Encoding bla CTX-M-15 in Pathogenic Escherichia coli in the Republic of Korea.

The emergence of third-generation cephalosporin resistance in Escherichia coli is increasing at an alarming rate in many countries. Thus, the aim of this study was to analyze co-infecting bla CTX-M-producing pathogenic E. coli isolates linked to three school outbreaks. Among 66 E. coli isolates, 44 were identified as ETEC O25, an ETEC isolate serotype was O2, and the other 21 were confirmed as EAEC O44. Interestingly, six patients were co-infected with EAEC O44 and ETEC O25. For these isolates, molecular analysis [antibiotic susceptibility testing, identification of the ß-lactamase gene, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)] was performed for further characterization. In addition, the transmission capacity of bla CTX-M genes was examined by conjugation experiments. Whole-genome sequencing (WGS) was performed on representative EAEC O44 and ETEC O25 isolates associated with co-infection and single-infection. All isolates were resistant to cefotaxime and ceftriaxone. All EAEC isolates carried the bla CTX-M-14 gene and all ETEC isolates the bla CTX-M-15 gene, as detected by multiplex PCR and sequencing analysis. Sequence type and PFGE results indicated three different patterns depending on the O serotype. WGS results of representative isolates revealed that the ETEC O25 strains harbored bla CTX-M-15 located on IncK plasmids associated with the Δbla TEM-bla CTX-M-15-orf477 transposon. The representative EAEC O44 isolates carried bla CTX-M-14 on the chromosome, which was surrounded by the ISEcp1-bla CTX-M-14-IS903 transposon. To the best of our knowledge, this is the first report of co-infection with chromosomally located bla CTX-M-14 and plasmid-encoding bla CTX-M-15 in pathogenic E. coli. Our findings indicate that resistance genes in clinical isolates can spread through concurrent combinations of chromosomes and plasmids.

Traveller-associated high-level ciprofloxacin-resistant Salmonella enterica Serovar Kentucky in the Republic of Korea.

OBJECTIVE: Salmonella enterica serotype Kentucky ST198 (S. Kentucky) is frequently associated with human infections and has been identified in travellers to North Africa, South Asia, Europe, and North America. The antimicrobial resistance of this serotype to multiple drugs, including ciprofloxacin (CIP), is a growing concern. However, little information is available regarding the occurrence and characterization of S. Kentucky in Korea. METHODS: To investigate the characteristics and possible origin of these infections, we characterized highly CIP-resistant S. Kentucky isolates collected through a national surveillance program. Single-nucleotide polymorphisms (SNPs) were identified by whole-genome sequencing (WGS), and genome sequencing was performed to investigate the genetic relationships and resistance mechanisms of the isolates. RESULTS: Ten CIP-resistant S. Kentucky strains were isolated from diarrheal patients in Korea from 2008 to 2017. The travel histories of the patients indicated that seven had returned from Southeast Asia. WGS of all the isolates revealed the presence of Salmonella genomic island 1 (SGI1) and substitutions in the gyrA and parC genes, which are known to confer resistance to CIP. A multilocus sequence type (MLST) analysis revealed that the isolates belonged to ST198, which has been prevalent in Europe and Africa. Furthermore, a phylogenetic analysis showed that all 10 isolates shared close genetic relationships. CONCLUSIONS: We report the identification of S. Kentucky in Korea through long-term surveillance. International travel, especially to Southeast Asia, has been a major risk factor for human infections of CIP-resistant S. Kentucky in Korea.

The role of international travellers in the spread of CTX-M-15-producing Shigella sonnei in the Republic of Korea.

BACKGROUND: Multidrug-resistant Shigella isolates have recently emerged as a serious public health threat worldwide. In particular, overseas travel is a risk factor for acquisition of antimicrobial-resistant Shigella strains. To explore the role of travel in the spread of cefotaxime-resistant Shigella sonnei in Korea, we screened 751 Shigella spp. isolates from 2007 to 2016 through the National Surveillance system, and 28 cephalosporin-resistant S. sonnei isolates were identified. METHODS: For cephalosporin-resistant S. sonnei isolates, epidemiological and molecular analyses (plasmid structure analysis, pulsed-field gel electrophoresis (PFGE) and high-quality single-nucleotide polymorphisms (hqSNPs) based on whole-genome sequencing (WGS)) were conducted to investigate the source of infection and transmission route. RESULTS: Among the 28 cefotaxime-resistant S. sonnei strains, 18 were isolated from travellers returning from Asia, including Vietnam (n=11). Molecular analysis of 18 blaCTX-M-type isolates revealed that 15 contain CTX-M-15; 50% of isolates from domestic patients contain CTX-M-14. Analysis of the genetic environments of the blaCTX-M-14 and blaCTX-M-15 genes revealed different genetic organization surrounding the blaCTX-M genes. Additionally, PFGE and hqSNP results suggested a large phylogenetic distance between the S. sonnei isolates related to overseas travel and those acquired domestically in Korea. CONCLUSION: Our study data demonstrates that two prevalent blaCTX-M genes, blaCTX-M-14 and blaCTX-M-15, have been circulating in S. sonnei in Korea over the last 10 years. Recently, international travellers are at a high risk for acquisition of CTX-M-15-producing S. sonnei in Korea.

Draft Genome Sequence of the First South Korean Clinical Isolate of Burkholderia pseudomallei, H0901.

We report here the draft genome sequence of Burkholderia pseudomallei H0901. This strain was isolated in 2003 from the first melioidosis patient in South Korea.

Construction of Luminescence- and Fluorescence-Tagged Burkholderia pseudomallei for Pathogen Tracking in a Mouse Model.

Molecular imaging is a powerful method for tracking various infectious disease-causing pathogens in host organisms. Currently, a dual molecular imaging method that can provide temporal and spatial information on infected hosts at the organism, organ, tissue, and cellular levels simultaneously has not been reported for Burkholderia pseudomallei, a high-risk pathogen that causes melioidosis. In this study, we have established an experimental method that provides spatiotemporal information on infected hosts using luminescent and fluorescent dual-labeled B. pseudomallei. Using this method, we visualized B. pseudomallei infection at the organism, organ, and tissue levels in a BALB/c mouse model by detecting its luminescence and fluorescence. The infection of B. pseudomallei at the cellular level was also visualized by its emitted fluorescence in infected macrophage cells. This method could be an extremely useful and applicable tool to study the pathogenesis of B. pseudomallei-related infectious diseases.

The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1ß, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.

Bacillus anthracis genomic DNA enhances lethal toxin-induced cytotoxicity through TNF-α production.

BACKGROUND: Bacillus anthracis is the etiological agent of anthrax. Lethal toxin (LT) produced by B. anthracis is a well-known key virulence factor for anthrax because of its strong cytotoxic activity. However, little is known about the role of B. anthracis genomic DNA (BAG) in anthrax pathogenesis. RESULTS: We examined the effect of BAG on TNF-α production and LT-mediated cytotoxicity during B. anthracis spore infection in mouse macrophage cell lines (RAW264.7 cells and J774A.1) and BALB/c mice. Infection of RAW264.7 cells with B. anthracis spores induced TNF-α expression in a multiplicity of infection (MOI)-dependent manner, and this enhancement was attenuated by the toll-like receptor (TLR) 9 inhibitor oligodeoxynucleotide (ODN)2088. BAG led to TNF-α expression in a dose- and time-dependent manner when applied to RAW264.7 cells. TNF-α expression induced by BAG was reduced by either pretreatment with TLR9 inhibitors (ODN2088 and chloroquine (CQ)) or transfection with TLR9 siRNA. Furthermore, BAG-induced TNF-α production in TLR9(+/+) macrophages was completely abrogated in TLR9(-/-) macrophages. BAG enhanced the phosphorylation of mitogen-activated protein kinases (MAPK), and BAG-induced TNF-α expression was attenuated by pretreatment with MAPK inhibitors. A reporter gene assay and confocal microscopy demonstrated that BAG increased NF-κB activation, which is responsible for TNF-α expression. Treatment with BAG alone showed no cytotoxic activity on the macrophage cell line J774A.1, whereas LT-mediated cytotoxicity was enhanced by treatment with BAG or TNF-α. Enhanced LT-induced lethality was also confirmed by BAG administration in mice. Furthermore, LT plus BAG-mediated lethality was significantly recovered by administration of Infliximab, an anti-TNF-α monoclonal antibody. CONCLUSIONS: Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

Monoclonal antibody against the poly-gamma-D-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge.

BACKGROUND: The poly-gamma-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. METHODS: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K(d) values of 0.8 microM and 2.6 microM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. RESULTS: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. GENERAL SIGNIFICANCE: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.

Complete genome sequence of Bacillus anthracis H9401, an isolate from a Korean patient with anthrax.

Bacillus anthracis H9401 (NCCP 12889) is an isolate from a Korean patient with gastrointestinal anthrax. The whole genome of H9401 was sequenced. It is a circular chromosome containing 5,480 open reading frames (ORFs) and two plasmids, pXO1 containing 202 ORFs and pXO2 containing 110 ORFs. H9401 shows high pathogenicity and genome sequence similarity to Ames Ancestor.

Expression and characterization of an iron-containing superoxide dismutase from Burkholderia pseudomallei.

A superoxide dismutase (SOD) gene from Burkholderia pseudomallei, the causative agent of melioidosis, was cloned and expressed in Escherichia coli, and its product was functionally and physically characterized. The gene has an open-reading frame of 579 bp. The deduced amino acid sequence has 192 residues with a calculated molecular mass of ~22 kDa. Sequence comparison with other bacterial SODs showed that the protein contains typical metal-binding motifs and other Fe-SOD-conserved residues. The sequence has substantial similarity with other bacterial Fe-SOD sequences. The enzymatic activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide or potassium cyanide, attributes that indeed are characteristic of typical bacterial Fe-SODs. Western blotting with antiserum against the recombinant Fe-SOD revealed that it is expressed in B. pseudomallei. Transformed E. coli that expressed the Fe-SOD had significantly increased SOD activity and was highly tolerant to paraquat-mediated replication inhibition, compared to transformed cells carrying an empty vector. Our results provide a basis for further biochemical characterization of the enzyme and elucidation of its role in the pathogenesis of B. pseudomallei.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis.

OBJECTIVE: Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. METHODS: Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. RESULTS: To examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. CONCLUSION: Neutralizing-antibody titers can be used as a surrogate marker.

The poly-γ-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity.

The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.