Emergence of genotype C1 Enterovirus A71 and its link with antigenic variation of virus in Taiwan.

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.

Prevalence of Type IV Pili-Mediated Twitching Motility in Streptococcus sanguinis Strains and Its Impact on Biofilm Formation and Host Adherence.

Type IV pili (Tfp) are known to mediate several biological activities, including surface-dependent twitching motility. Although a pil gene cluster for Tfp biosynthesis is found in all sequenced Streptococcus sanguinis strains, Tfp-mediated twitching motility is less commonly detected. Upon examining 81 clinical strains, 39 strains generated twitching zones on blood agar plates (BAP), while 27 strains displayed twitching on Todd-Hewitt (TH) agar. Although BAP appears to be more suitable for the development of twitching zones, 5 strains exhibited twitching motility only on TH agar, indicating that twitching motility is not only strain specific but also sensitive to growth media. Furthermore, different twitching phenotypes were observed in strains expressing comparable levels of pilT, encoding the retraction ATPase, suggesting that the twitching phenotype on agar plates is regulated by multiple factors. By using a PilT-null and a pilin protein-null derivative (CHW02) of twitching-active S. sanguinis CGMH010, we found that Tfp retraction was essential for biofilm stability. Further, biofilm growth was amplified in CHW02 in the absence of shearing force, indicating that S. sanguinis may utilize other ligands for biofilm formation in the absence of Tfp. Similar to SK36, Tfp from CGMH010 were required for colonization of host cells, but PilT only marginally affected adherence and only in the twitching-active strain. Taken together, the results suggest that Tfp participates in host cell adherence and that Tfp retraction facilitates biofilm stability. IMPORTANCE Although the gene clusters encoding Tfp are commonly present in Streptococcus sanguinis, not all strains express surface-dependent twitching motility on agar surfaces. Regardless of whether the Tfp could drive motility, Tfp can serve as a ligand for the colonization of host cells. Though many S. sanguinis strains lack twitching activity, motility can enhance biofilm stability in a twitching-active strain; thus, perhaps motility provides little or no advantage to the survival of bacteria within dental plaque. Rather, Tfp retraction could provide additional advantages for the bacteria to establish infections outside the oral cavity.

Functional Analysis of a Fibronectin Binding Protein of Streptococcus parasanguinis FW213.

Streptococcus parasanguinis is a primary colonizer of dental plaque and an opportunistic pathogen for subacute endocarditis. A putative fibronectin binding protein (Spaf_1409) that lacks both an N-terminal signal peptide and a C-terminal cell wall-anchoring motif was identified from the S. parasanguinis FW213 genome. Spaf_1409 was abundantly present in the cytoplasm and also was found in the cell wall preparation and culture supernatant. By using an isogenic mutant strain, MPH4, Spaf_1409 was found to mediate the binding of S. parasanguinis FW213 to fibronectin. Inactivation of Spaf_1409 did not significantly alter the mass of static biofilm, but reduced the resistance of S. parasanguinis against the shearing force in a flow cell biofilm system, resulting in scattered biofilm. The mortality in Galleria mellonella larvae infected with MPH4 was higher than in those infected with wild-type S. parasanguinis. However, fewer viable bacterial cells were recovered from larvae infected with MPH4, compared to those infected with wild-type S. parasanguinis, up to 42 h post infection, suggesting that the infection by MPH4, but not the growth, was responsible for the elevated mortality. The phagocytic analysis using flow cytometry indicated that Spaf_1409 participates in the recognition of S. parasanguinis FW213 by RAW264.7 macrophages, suggesting that inactivation of Spaf_1409 intensified the immune responses in larvae, leading to larval death. Taken together, the data indicate that Spaf_1409 plays different roles in the development of dental biofilm and in systemic infections.

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36.

Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5' to the cluster and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5' promoter, which is located more than 800 bases 5' to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hairlike surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313 to SSA_2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes downregulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation.IMPORTANCE The proteins and assembly machinery of the type IV pili (Tfp) are conserved throughout bacteria and archaea, and yet the function of this surface structure differs from species to species and even from strain to strain. As seen in Streptococcus sanguinis SK36, the expression of the Tfp gene cluster results in a hairlike surface structure that is much shorter than the typical Tfp. This pilus is essential for the adherence of SK36 but is not involved in motility. Being a member of the highly diverse dental biofilm, perhaps S. sanguinis could more effectively utilize this structure to adhere to host cells and to interact with other microbes within the same niche.

YMC-2011, a Temperate Phage of Streptococcus salivarius 57.I.

Streptococcus salivarius is an abundant isolate of the oral cavity. The genome of S. salivarius 57.I consists of a 2-Mb chromosome and a 40,758-bp circular molecule, designated YMC-2011. Annotation of YMC-2011 revealed 55 open reading frames, most of them associated with phage production, although plaque formation is not observed in S. salivarius 57.I after lytic induction using mitomycin C. Results from Southern hybridization and quantitative real-time PCR confirmed that YMC-2011 exists extrachromosomally, with an estimated copy number of 3 to 4. Phage particles were isolated from the supernatant of mitomycin C-treated S. salivarius 57.I cultures, and transmission electron microscopic examination indicated that YMC-2011 belongs to the Siphoviridae family. Phylogenetic analysis suggests that phage YMC-2011 and the cos-type phages of Streptococcus thermophilus originated from a common ancestor. An extended -10 element (p L ) and a σ70-like promoter (p R ) were mapped 5' to Ssal_phage00013 (encoding a CI-like repressor) and Ssal_phage00014 (encoding a hypothetical protein), respectively, using 5' rapid amplification of cDNA ends, indicating that YMC-2011 transcribes at least two mRNAs in opposite orientations. Studies using promoter-chloramphenicol acetyltransferase reporter gene fusions revealed that p R , but not p L , was sensitive to mitomycin C induction, suggesting that the switch from lysogenic growth to lytic growth was controlled mainly by the activity of these two promoters. In conclusion, a lysogenic state is maintained in S. salivarius 57.I, presumably by the repression of genes encoding proteins for lytic growth.IMPORTANCE The movement of mobile genetic elements such as bacteriophages and the establishment of lysogens may have profound effects on the balance of microbial ecology where lysogenic bacteria reside. The discovery of phage YMC-2011 from Streptococcus salivarius 57.I suggests that YMC-2011 and Streptococcus thermophilus-infecting phages share an ancestor. Although S. salivarius and S. thermophilus are close phylogenetically, S. salivarius is a natural inhabitant of the human mouth, whereas S. thermophilus is commonly found in the mammary mucosa of bovine species. Thus, the identification of YMC-2011 suggests that horizontal gene transfer via phage infection could take place between species from different ecological niches.

Metabolomics analysis reveals changes related to pseudocyst formation induced by iron depletion in Trichomonas vaginalis.

BACKGROUND: Iron is an essential element for cellular functions, such as energy metabolism. Trichomonas vaginalis, a human urogenital tract pathogen, is capable of surviving in the environment without sufficient iron supplementation. Pseudocysts (cyst-like structures) are an environmentally tolerated stage of this parasite while encountering undesired conditions, including iron deficiency. We previously demonstrated that iron deficiency induces more active glycolysis but a drastic downregulation of hydrogenosomal energy metabolic enzymes. Therefore, the metabolic direction of the end product of glycolysis is still controversial. METHODS: In the present work, we conducted an LC‒MS-based metabolomics analysis to obtain accurate insights into the enzymatic events of T. vaginalis under iron-depleted (ID) conditions. RESULTS: First, we showed the possible digestion of glycogen, cellulose polymerization, and accumulation of raffinose family oligosaccharides (RFOs). Second, a medium-chain fatty acid (MCFA), capric acid, was elevated, whereas most detected C18 fatty acids were reduced significantly. Third, amino acids were mostly reduced, especially alanine, glutamate, and serine. Thirty-three dipeptides showed significant accumulation in ID cells, which was probably associated with the decrease in amino acids. Our results indicated that glycogen was metabolized as the carbon source, and the structural component cellulose was synthesized at same time. The decrease in C18 fatty acids implied possible incorporation in the membranous compartment for pseudocyst formation. The decrease in amino acids accompanied by an increase in dipeptides implied incomplete proteolysis. These enzymatic reactions (alanine dehydrogenase, glutamate dehydrogenase, and threonine dehydratase) were likely involved in ammonia release. CONCLUSION: These findings highlighted the possible glycogen utilization, cellulose biosynthesis, and fatty acid incorporation in pseudocyst formation as well as NO precursor ammonia production induced by iron-depleted stress.

Functional Analysis of the Collagen Binding Proteins of Streptococcus parasanguinis FW213.

Streptococcus parasanguinis is a dominant isolate of dental plaque and an opportunistic pathogen associated with subacute endocarditis. As the expression of collagen binding proteins (CBPs) could promote the establishment of S. parasanguinis in the host, the functions of three putative CBP-encoding loci, Spaf_0420, Spaf_1570, and Spaf_1573, were analyzed using isogenic mutant strains. It was revealed that S. parasanguinis FW213 bound effectively to fibronectin and type I collagen, but the strain's affinity for laminin and type IV collagen was quite low. By using various deletion derivatives, it was found that these three loci mediated the binding of S. parasanguinis to multiple extracellular matrix molecules, with type I collagen as the common substrate. Derivative strains with a deletion in any of the three loci expressed reduced binding to trypsin-treated swine heart valves. The deletion of these loci also reduced the viable count of S. parasanguinis bacteria within macrophages, especially the loss of Spaf_0420, but only strains with deletions in Spaf_0420 and Spaf_1570 expressed reduced virulence in the Galleria mellonella larva model. The deletion of Spaf_1570 and Spaf_1573 affected mainly the structure, but not the overall mass, of biofilm cultures in a flow cell system. Thus, CBPs are likely to be more critical for the initial colonization of S. parasanguinis on host tissues during the development of endocarditis.IMPORTANCE Bacteria generally can utilize multiple adhesins to establish themselves in the host. We found that Streptococcus parasanguinis, a dominant oral commensal and an opportunistic pathogen for subacute endocarditis, possesses at least three collagen-binding proteins that enable S. parasanguinis to successfully colonize damaged heart tissues and escape innate immune clearance. The binding specificities of these three proteins for extracellular matrix molecules differ, although all three proteins participate in biofilm formation by S. parasanguinis The "multiligand for multisubstrate" feature of these adhesins may explain the high adaptability of this microbe to different tissue sites.

Epitope-associated and specificity-focused features of EV71-neutralizing antibody repertoires from plasmablasts of infected children.

Protective antibody levels are critical for protection from severe enterovirus 71 infection. However, little is known about the specificities and functional properties of the enterovirus 71-specific antibodies induced by natural infection in humans. Here we characterize 191 plasmablast-derived monoclonal antibodies from three enterovirus 71-infected children, each of whom shows a distinct serological response. Of the 84 enterovirus 71-specific antibodies, neutralizing antibodies that target the rims and floor of the capsid canyon exhibit broad and potent activities at the nanogram level against viruses isolated in 1998-2016. We also find a subset of infected children whose enterovirus 71-specific antibodies are focused on the 3- and 2-fold plateau epitopes localized at the margin of pentamers, and this type of antibody response is associated with lower serum titers against recently circulating strains. Our data provide new insights into the enterovirus 71-specific antibodies induced by natural infection at the serological and clonal levels.Enterovirus 71 is a leading cause of hand-foot-and-mouth disease and herpangina. Here, the authors characterize a large panel of plasmablast-derived IgG mAbs that target the capsid of EV71 to identify neutralizing antibodies induced by natural infection.

Bedside immunochromatographic test for enterovirus 71 infection in children.

BACKGROUND: Enterovirus 71 (EV71) causes frequent outbreaks worldwide, particularly in the Asia-Pacific area. Its quick spread is a critical challenge for public health and timely preventive measures and clinical management therefore rely on early detection. There is a need for a rapid, easy-to-use, and reliable method for detecting EV71 infections. OBJECTIVE: The study aimed to evaluate a bedside immunochromatography (ICT) kit for diagnosing acute EV71 infection in children. STUDY DESIGN: Pediatric patients with herpangina or hand-foot-mouth disease were randomly and prospectively enrolled from hospitals across Taiwan. Throat or rectal swabs were collected for viral culture and reverse-transcriptase polymerase chain reaction (RTPCR). For the ICT kit, whole blood was obtained by ear piercing, finger-sticking, or venipuncture. The results of ICT, virus isolation and RTPCR in clinical samples were compared. RESULTS: Of the 156 patients enrolled, 91 (58%), 64 (41%) and 72 (46%) had positive results of the ICT kit, viral culture and RTPCR, respectively. Laboratory-confirmed infection with either positive EV71 culture or RTPCR was used as the diagnostic standard. The sensitivity and specificity of the ICT kit was 84% and 77%, respectively. The viral culture and RTPCR had relatively lower sensitivity but higher specificity. The patient's age did not affect the performance of the ICT, viral culture and RTPCR. However, a low sensitivity of ICT kit was noted before the second day of disease onset. CONCLUSIONS: The ICT kit may serve as a simple, quick and reliable method for the bedside diagnosis of acute EV71 infection in children.

Complete genome and transcriptomes of Streptococcus parasanguinis FW213: phylogenic relations and potential virulence mechanisms.

Streptococcus parasanguinis, a primary colonizer of the tooth surface, is also an opportunistic pathogen for subacute endocarditis. The complete genome of strain FW213 was determined using the traditional shotgun sequencing approach and further refined by the transcriptomes of cells in early exponential and early stationary growth phases in this study. The transcriptomes also discovered 10 transcripts encoding known hypothetical proteins, one pseudogene, five transcripts matched to the Rfam and additional 87 putative small RNAs within the intergenic regions defined by the GLIMMER analysis. The genome contains five acquired genomic islands (GIs) encoding proteins which potentially contribute to the overall pathogenic capacity and fitness of this microbe. The differential expression of the GIs and various open reading frames outside the GIs at the two growth phases suggested that FW213 possess a range of mechanisms to avoid host immune clearance, to colonize host tissues, to survive within oral biofilms and to overcome various environmental insults. Furthermore, the comparative genome analysis of five S. parasanguinis strains indicates that albeit S. parasanguinis strains are highly conserved, variations in the genome content exist. These variations may reflect differences in pathogenic potential between the strains.

Clinical and epidemiologic features of Coxsackievirus A6 infection in children in northern Taiwan between 2004 and 2009.

BACKGROUND: Isolates of Coxsackievirus A6 (Cox A6) is increasing clinically in 2009 in Taiwan but detailed clinical features of Cox A6 infections in children have not been reported. This study is to define clinical manifestations and laboratory findings of Cox A6 infection in children. METHODS: From January 2004 to December 2009, a total of 4,664 children with enterovirus infections, based on throat virus culture, were treated in Chang Gung Children's hospital. Two hundred and ninety-six (6.3%) patients positive for Cox A6 infection were included in this study. One hundred and forty-one (47.6%) inpatients were further analyzed for clinical presentations, laboratory findings, and clinical diagnoses. RESULTS: There were two peaks of Cox A6 infection in 2007 and 2009 during the study period, especially during the warm season. The proportion of Cox A6 among total enterovirus isolates was 15.5% in 2007 and up to 22.2% in 2009. The mean age of inpatients was 2.42 ± 0.14 years. The mean hospitalization duration was 4.21 ± 0.11 days. The most common symptoms were fever (100%), oral ulcers (90.8%), and decreased oral intake (89.4%). The mean duration of fever was 2.78 ± 1 days (range, 1-7 days). Seventy-seven (54.6%) patients had fever more than 3 days. The mean leukocyte count was 14,850/mm(3), and 63 (45%) patients had leukocytosis (>15,000/mm(3)). The mean serum C-reactive protein (CRP) level was 44.1 ± 3.3 mg/L (normal, <10 mg/L) and 62 (44%) had a CRP level >40 mg/L. One hundred and eight (76.6%) inpatients were diagnosed as herpangina and 18 (12.8%) hand-foot-mouth disease. Three patients had complications, including aseptic meningitis in one and encephalitis in two. All 141 inpatients recovered uneventfully. CONCLUSIONS: Cox A6 is among the major serotypes of enteroviruses in Taiwan and most cases presented as herpangina and hand-foot-mouth disease. Nearly half of the cases may have leukocytosis and elevated CRP levels. Outcomes are usually good.

Comparison of clinical features between coxsackievirus A2 and enterovirus 71 during the enterovirus outbreak in Taiwan, 2008: a children's hospital experience.

BACKGROUND/PURPOSE: Coxsackievirus A2 (Cox A2) was the predominant serotype in the enterovirus outbreak in Taiwan, 2008. However, detailed clinical features of Cox A2 infection have not been reported. In this study, we compared Cox A2 with enterovirus 71 (EV71) in terms of clinical manifestation and epidemiology during the 2008 enterovirus outbreak in Taiwan. METHODS: A total of 280 hospitalized patients (97 with culture-proven EV71 infection and 183 with culture-proven Cox A2 infection) in 2008 at the Chang Gung Children's Medical Center were enrolled in this study. Epidemiologic data, clinical manifestations, and outcomes for these patients were collected and compared. RESULTS: Both Cox A2 and EV71 serotypes peaked in June and declined soon afterwards. Seventy-one percent of the patients were younger than 3 years of age. Both groups had the same male-to-female ratio of 1.6:1. Patients with EV71 infection had a significantly longer hospitalization period (4.1 vs. 3.0 days, p< 0.001). Fever, fever for more than 3 days with a temperature above 39 degrees C, lethargy, poor activity, poor appetite and a myoclonic jerk were significantly associated with EV71 infection. Fever, or fever with a temperature above 39 degrees C, febrile seizure, elevated white cell counts, and elevated serum C-reactive protein concentrations were significantly associated with Cox A2 infection. Most patients with EV71 infection presented with hand-foot-mouth disease (78.3%), while most Cox A2-infected patients presented with herpangina (83.6%). Central nervous system complications were found in 18.6% of EV71-infected children, but only in 1.1% of Cox A2-infected children. All the patients with Cox A2 infection showed total recovery. One patient with EV71 infection died from encephalitis with cardiopulmonary failure, and 6.2% of EV71-infected children had neurologic sequelae. CONCLUSION: Both Cox A2 and EV71 serotypes accounted for the enterovirus outbreak in Taiwanese children in 2008. Compared with those infected by EV71, the children with Cox A2 infection mostly presented with herpangina, had fewer central nervous system complications, and had better overall outcome.

Fulminant septicaemia caused by multi-drug-resistant Streptococcus mitis following unrelated cord blood transplantation.

Streptococcus mitis (a common and usually harmless bacterium found in the nose, mouth and throat) can have an unusually high level of resistance to beta-lactam antibiotics. We report a patient who developed fatal Streptococcus mitis septicaemia following unrelated cord blood transplantation. Administration of vancomycin to patients with recurrent fever during allogeneic stem cell transplantation might be indicated.