Interplay between group A Streptococcus and host innate immune responses.

SUMMARYGroup A Streptococcus (GAS), also known as Streptococcus pyogenes, is a clinically well-adapted human pathogen that harbors rich virulence determinants contributing to a broad spectrum of diseases. GAS is capable of invading epithelial, endothelial, and professional phagocytic cells while evading host innate immune responses, including phagocytosis, selective autophagy, light chain 3-associated phagocytosis, and inflammation. However, without a more complete understanding of the different ways invasive GAS infections develop, it is difficult to appreciate how GAS survives and multiplies in host cells that have interactive immune networks. This review article attempts to provide an overview of the behaviors and mechanisms that allow pathogenic GAS to invade cells, along with the strategies that host cells practice to constrain GAS infection. We highlight the counteractions taken by GAS to apply virulence factors such as streptolysin O, nicotinamide-adenine dinucleotidase, and streptococcal pyrogenic exotoxin B as a hindrance to host innate immune responses.

A 19-year longitudinal study to characterize carbapenem-nonsusceptible Acinetobacter isolated from patients with bloodstream infections and the contribution of conjugative plasmids to carbapenem resistance and virulence.

BACKGROUND: This study aimed to characterize carbapenem-nonsusceptible Acinetobacter (CNSA) isolated from patients with bacteremia from 1997 to 2015. METHODS: A total of 173 CNSA (12.3%) was recovered from 1403 Acinetobacter isolates. The presence of selected ß-lactamase genes in CNSA was determined by PCR amplification. The conjugation test was used to determine the transferability of metallo-ß-lactamase (MBL)-carrying plasmids. Whole genome sequencing in combination with phenotypic assays was carried out to characterize MBL-plasmids. RESULTS: In general, a trend of increasing numbers of CNSA was observed. Among the 173 CNSA, A. baumannii (54.9%) was the most common species, followed by A. nosocomialis (23.1%) and A. soli (12.1%). A total of 49 (28.3%) CNSA were extensively drug-resistant, and all were A. baumannii. The most common class D carbapenemase gene in 173 CNSA was blaOXA-24-like (32.4%), followed by ISAba1-blaOXA-51-like (20.8%), ISAba1-blaOXA-23 (20.2%), and IS1006/IS1008-blaOXA-58 (11.6%). MBL genes, blaVIM-11,blaIMP-1, and blaIMP-19 were detected in 9 (5.2%), 20 (11.6%), and 1 (0.6%) CNSA isolates, respectively. Transfer of MBL genes to AB218 and AN254 recipient cells was successful for 7 and 6 of the 30 MBL-plasmids, respectively. The seven AB218-derived transconjugants carrying MBL-plasmids produced less biofilm but showed higher virulence to larvae than recipient AB218. CONCLUSIONS: Our 19-year longitudinal study revealed a stable increase in CNSA during 2005-2015. blaOXA-24-like, ISAba1-blaOXA-51-like, and ISAba1-blaOXA-23 were the major determinants of Acinetobacter carbapenem resistance. MBL-carrying plasmids contribute not only to the carbapenem resistance but also to A. baumannii virulence.

Longitudinal neutralizing antibody responses after SARS-CoV-2 infection: A convalescent cohort study in Taiwan.

BACKGROUND: Understanding the neutralizing antibody (NAb) titer against COVID-19 over time is important to provide information for vaccine implementation. The longitudinal NAb titer over one year after SARS-CoV-2 infection is still unclear. The purposes of this study are to evaluate the duration of the neutralizing NAb titers in COVID-19 convalescents and factors associated with the titer positive duration. METHODS: A cohort study followed COVID-19 individuals diagnosed between 2020 and 2021 May 15th from the COVID-19 database from the Taiwan Centers for Disease Control. We analyzed NAb titers from convalescent SARS-CoV-2 individuals. We used generalized estimating equations (GEE) and a Cox regression model to summarize the factors associated with NAb titers against COVID-19 decaying in the vaccine-free population. RESULTS: A total of 203 convalescent subjects with 297 analytic samples were followed for a period of up to 588 days. Our study suggests that convalescent COVID-19 in individuals after more than a year and four months pertains to only 25% of positive titers. The GEE model indicates that longer follow-up duration was associated with a significantly lower NAb titer. The Cox regression model indicated the disease severity with advanced condition was associated with maintaining NAb titers (adjusted hazard ratio: 2.01, 95% CI: 1.11-3.63) and that smoking was also associated with higher risk of negative NAb titers (adjusted hazard ratio: 0.55, 95% CI: 0.33-0.92). CONCLUSIONS: Neutralizing antibody titers diminished after more than a year. The antibody titer response against SARS-CoV-2 in naturally convalescent individuals provides a reference for vaccinations.

Structural basis underlying the synergism of NADase and SLO during group A Streptococcus infection.

Group A Streptococcus (GAS) is a strict human pathogen possessing a unique pathogenic trait that utilizes the cooperative activity of NAD+-glycohydrolase (NADase) and Streptolysin O (SLO) to enhance its virulence. How NADase interacts with SLO to synergistically promote GAS cytotoxicity and intracellular survival is a long-standing question. Here, the structure and dynamic nature of the NADase/SLO complex are elucidated by X-ray crystallography and small-angle scattering, illustrating atomic details of the complex interface and functionally relevant conformations. Structure-guided studies reveal a salt-bridge interaction between NADase and SLO is important to cytotoxicity and resistance to phagocytic killing during GAS infection. Furthermore, the biological significance of the NADase/SLO complex in GAS virulence is demonstrated in a murine infection model. Overall, this work delivers the structure-functional relationship of the NADase/SLO complex and pinpoints the key interacting residues that are central to the coordinated actions of NADase and SLO in the pathogenesis of GAS infection.

VEGF-Mediated Augmentation of Autophagic and Lysosomal Activity in Endothelial Cells Defends against Intracellular Streptococcus pyogenes.

Group A Streptococcus (GAS), a deleterious human-pathogenic bacterium, causes life-threatening diseases such as sepsis and necrotic fasciitis. We recently reported that GAS survives and replicates within blood vessel endothelial cells because these cells are intrinsically defective in xenophagy. Because blood vessel endothelial cells are relatively germfree environments, specific stimulation may be required to sufficiently induce xenophagy. Here, we explored how vascular endothelial growth factor (VEGF) promoted xenophagy and lysosomal activity in endothelial cells. These effects were achieved by amplifying the activation of TFEB, a transcriptional factor crucial for lysosome/autophagy biogenesis, via cAMP-mediated calcium release. In a mouse model of local infection with GAS, the VEGF level was significantly elevated at the infection site. Interestingly, low serum VEGF levels were found in a mouse model of invasive bacteremia and in patients with severe GAS-induced sepsis. Moreover, the administration of VEGF improved the survival of GAS-infected mice. We propose a novel theory regarding GAS infection in endothelial cells, wherein VEGF concentrations in the systemic circulation play a critical role. IMPORTANCE Sepsis caused by Streptococcus pyogenes is a life-threatening condition. Blood vessel endothelial cells should serve as a barrier to infection, although we recently reported that endothelial cells allow intracellular GAS proliferation due to defective xenophagy. In this study, we revealed that administration of VEGF augmented both xenophagy and lysosomal activity in these cells, leading to the efficient killing of intracellular GAS. By comparison, the opposite relationship was observed in vivo, as low serum VEGF concentrations were accompanied by high-severity sepsis in both a mouse model and in human patients. Administration of VEGF reduced mortality in the GAS sepsis model. Based on these findings, we hypothesize that during acute infection, strong VEGF stimulation boosts the intracellular defense system of the endothelium to provide a stronger blood vessel barrier, thereby helping to prevent bacterial dissemination.

Uremic Toxin-Producing Bacteroides Species Prevail in the Gut Microbiota of Taiwanese CKD Patients: An Analysis Using the New Taiwan Microbiome Baseline.

Rationale and Objective: Gut microbiota have been targeted by alternative therapies for non-communicable diseases. We examined the gut microbiota of a healthy Taiwanese population, identified various bacterial drivers in different demographics, and compared them with dialysis patients to associate kidney disease progression with changes in gut microbiota. Study Design: This was a cross-sectional cohort study. Settings and Participants: Fecal samples were obtained from 119 healthy Taiwanese volunteers, and 16S rRNA sequencing was done on the V3-V4 regions to identify the bacterial enterotypes. Twenty-six samples from the above cohort were compared with fecal samples from 22 peritoneal dialysis and 16 hemodialysis patients to identify species-level bacterial biomarkers in the dysbiotic gut of chronic kidney disease (CKD) patients. Results: Specific bacterial species were identified pertaining to different demographics such as gender, age, BMI, physical activity, and sleeping habits. Dialysis patients had a significant difference in gut microbiome composition compared to healthy controls. The most abundant genus identified in CKD patients was Bacteroides, and at the species level hemodialysis patients showed significant abundance in B. ovatus, B. caccae, B. uniformis, and peritoneal dialysis patients showed higher abundance in Blautia producta (p ≤ 0.05) than the control group. Pathways pertaining to the production of uremic toxins were enriched in CKD patients. The abundance of the bacterial species depended on the type of dialysis treatment. Conclusion: This study characterizes the healthy gut microbiome of a Taiwanese population in terms of various demographics. In a case-control examination, the results showed the alteration in gut microbiota in CKD patients corresponding to different dialysis treatments. Also, this study identified the bacterial species abundant in CKD patients and their possible role in complicating the patients' condition.

Association between Escherichia coli with NotI-restriction resistance and urinary tract infections.

BACKGROUND: Escherichia coli is the most common cause of urinary tract infections (UTIs). It is widely accepted that uropathogenic E. coli (UPEC) mainly emerge from the distal gut microbiota. Identification of bacterial characteristics that are able to differentiate UPEC from fecal commensal strains will facilitate the development of novel strategies to detect and monitor the spread of UPEC. METHODS: Fifty fecal commensal, 83 UTI-associated and 40 biliary tract infection (BTI)-associated E. coli isolates were analyzed. The NotI restriction patterns of chromosomal DNA in the isolates were determined by pulse-field gel electrophoresis. The phylogenetic types and the presence of 9 known virulence genes of each isolate were determined by PCR analyses. Additionally, the susceptibilities of the isolates to antibiotics were revealed. Then the associations of NotI resistance with UTI-associated isolates, phylotypes, and antibiotic resistance were assessed. RESULTS: NotI resistance was correlated with UTI-associated isolates, compared to the fecal isolates. Consistently, NotI-resistant isolates harbored a greater number of virulence factors and mainly belonged to phylotype B2. Additionally NotI resistance was correlated with chloramphenicol resistance among the bacteria. Among the fecal, UTI-associated and BTI-associated groups, the distribution of NotI-resistant group B2 isolates was correlated with UTI-associated bacteria. CONCLUSION: NotI resistance alone is a potential marker for distinguishing fecal strains and UPEC, while the combination of NotI resistance and B2 phylogeny is a candidate marker to differentiate UPEC from fecal and other extraintestinal pathogenic E. coli. Additionally, NotI resistance may be valuable for assessing the potential of chloramphenicol resistance of E. coli.

Escherichia coli FimH adhesins act synergistically with PapGII adhesins for enhancing establishment and maintenance of kidney infection.

BACKGROUND: FimH adhesin is proposed to enhance Escherichia coli kidney infection by acting with PapGII adhesin, but genetic epidemiology study and animal study have not been widely conducted to confirm this hypothesis. METHODS: We compared the prevalence of adhesin gene and their coexistent pattern between upper and lower urinary tract infection (UTI) strains. fimH mutant (EC114FM), papGII mutant (EC114PM) and fimH/papGII double mutant (EC114DM) were constructed from a pylonephritogenic strain (EC114). We compared among these strains for the infection ability in bladders and kidneys of female BALB/c mice challenged transurethrally with these bacteria and assessed 1, 3, and 7 days after inoculation. RESULTS: Strains carrying fimH-only genotype were significantly more prevalent in lower UTI (P < 0.001). Strains carrying the fimH/papGII, but not papGII-only, were significantly associated with upper UTI (P = 0.001). Incidence of kidney infection increased after inoculation with EC114 on days 1 and 3, at both low and high dose, as compared with EC114DM; and the effect was greater than the sum of individual effect of EC114PM and EC114FM. Geometric means of quantitative bacterial counts in the kidneys significantly decreased when challenged with EC114FM on days 3 and 7, EC114PM on day 3 and EC114DM on day 1 after inoculation at high dose, as compared with EC114 (all P < 0.05). CONCLUSIONS: We confirmed the advantage and synergistic action of FimH and PapGII for E. coli kidney infection and concluded that antagonists against FimH and PapGII adhesin may prevent kidney infection and enable its management.

The Ferric Citrate Uptake System Encoded in a Novel bla CTX-M-3- and bla TEM-1-Harboring Conjugative Plasmid Contributes to the Virulence of Escherichia coli.

Escherichia coli is one major cause of bacterial infections and can horizontally acquire antimicrobial resistance and virulence genes through conjugation. Because conjugative plasmids can rapidly spread among bacteria of different species, the plasmids carrying both antimicrobial resistance and virulence genes may pose a significant threat to public health. Therefore, the identification and characterization of these plasmids may facilitate a better understanding of E. coli pathogenesis and the development of new strategies against E. coli infections. Because iron uptake ability is a potential virulence trait of bacteria, we screened for E. coli conjugative plasmids able to confer both iron uptake ability and ampicillin resistance. The plasmid pEC41, which was derived from the bacteremia clinical isolate EC41, was identified. EC41, which carried the fimH27 allele, belonged to sequence type (ST) 405 and phylogroup D. According to the sequencing analyses, pEC41 was 86 kb in size, and its backbone structure was almost identical to that of another highly conjugative plasmid, pCTX-M3, in which the extended-spectrum ß-lactamase gene bla CTX-M-3 was originally identified. pEC41 carried bla CTX-M-3 and bla TEM-1. The ferric citrate uptake (fec) system was identified in pEC41 and was responsible for conferring iron uptake ability. The fec system contributes to the pathogenesis of EC41 in systemic infections but not in urinary tract infections (UTIs). However, this system promoted competitive fitness of a cystitis-associated clinical isolate to colonize urinary tracts. Additionally, the distribution of the fec system was related to E. coli isolates associated with human bacteremia and UTIs. In summary, the present study identified a novel conjugative plasmid, pEC41, which conferred both antimicrobial resistance and an extra iron uptake ability to E. coli. The iron uptake ability was encoded in the fec system and contributed to E. coli pathogenesis. This study is the first to show that the fec system is a virulence factor in E. coli.

Glycosyltransferase Jhp0106 (PseE) contributes to flagellin maturation in Helicobacter pylori.

BACKGROUND: Flagella-mediated motility is both a crucial virulence determinant of Helicobacter pylori and a factor associated with gastrointestinal diseases. Flagellar formation requires flagellins to be glycosylated with pseudaminic acid (Pse), a process that has been extensively studied. However, the transfer of Pse to flagellins remains poorly understood. Therefore, the aim of this study is to characterize a putative glycosyltransferase jhp0106 in flagellar formation. MATERIALS AND METHODS: Western blotting and chemical deglycosylation were performed to examine FlaA glycosylation. Protein structural analyses were executed to identify the active site residues of Jhp0106, while the Jhp0106-FlaA interaction was examined using a bacterial two-hybrid assay. Lastly, site-directed mutants with mutated active site residues in the jhp0106 gene were generated and investigated using a motility assay, Western blotting, cDNA-qPCR analysis, and electron microscopic examination. RESULTS: Loss of flagellar formation in the Δjhp0106 mutant was confirmed to be associated with non-glycosylated FlaA. Furthermore, three active site residues of Jhp0106 (S350, F376, and E415) were identified within a potential substrate-binding region. The interaction between FlaA and Jhp0106, Jhp0106::S350A, Jhp0106::F376A, or Jhp0106::E415A was determined to be significant. As well, the substitution of S350A, F376A, or E415A in the site-directed Δjhp0106 mutants resulted in impaired motility, deficient FlaA glycosylation, and lacking flagella. However, these phenotypic changes were regardless of flaA expression, implying an indefinite proteolytic degradation of FlaA occurred. CONCLUSIONS: This study demonstrated that Jhp0106 (PseE) binds to FlaA mediating FlaA glycosylation and flagellar formation. Our discovery of PseE has revealed a new glycosyltransferase family responsible for flagellin glycosylation in pathogens.

Emergence of macrolide-resistant Streptococcus pyogenes emm12 in southern Taiwan from 2000 to 2019.

BACKGROUND: Group A Streptococcus (GAS) is an important pathogen causing morbidity and mortality worldwide. Surveillance of resistance and emm type has important implication to provide helpful information on the changing GAS epidemiology and empirical treatment. METHODS: To study the emergence of resistant GAS in children with upper respiratory tract infection (URTI), a retrospective study was conducted from 2000 to 2019 in southern Taiwan. Microbiological studies, including antibiotic susceptibility, were performed. GAS emm types and sequences were determined by molecular methods. The population was divided into two separate decades to analyze potential changes over time. The 1st decade was 2000-2009; the 2nd decade was 2010-2019. Multivariate analyses were performed to identify independent risk factors associated with macrolide resistance between these periods. RESULTS: A total of 320 GAS from 339 children were enrolled. Most of the children (75%) were under 9 years of age. The most common diagnosis was scarlet fever (225, 66.4%), and the frequency increased from 54.8% in the 1st to 77.9% in the 2nd decade (p < 0.0001). There was a significant increase in resistance to erythromycin and azithromycin from 18.1%, 19.3% in the 1st to 58.4%, 61.0% in the 2nd decade (p < 0.0001). This was associated with clonal expansion of the GAS emm12-ST36 which carrying erm(B) and tet(M) from 3.0% in the 1st to 53.2% in the 2nd decade (p < 0.0001). CONCLUSIONS: Significant emergence of macrolide-resistant GAS emm12-ST36 in children supports the need for continuing surveillance and investigation for the clonal virulence.

Nicotinamide Increases Intracellular NAD+ Content to Enhance Autophagy-Mediated Group A Streptococcal Clearance in Endothelial Cells.

Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD+ level and cell viability were respectively measured by NAD+ quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD+ content and NAD+/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or S. Typhimurium in endothelial cells. These results indicate that intracellular NAD+ homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.

The role of the bacterial protease Prc in the uropathogenesis of extraintestinal pathogenic Escherichia coli.

BACKGROUND: Extraintestinal pathogenic E. coli (ExPEC) remains one of the most prevalent bacterial pathogens that cause extraintestinal infections, including neonatal meningitis, septicemia, and urinary tract (UT) infections (UTIs). Antibiotic therapy has been the conventional treatment for such infections, but its efficacy has decreased due to the emergence of antibiotic-resistant bacteria. Identification and characterization of bacterial factors that contribute to the severity of infection would facilitate the development of novel therapeutic strategies. The ExPEC periplasmic protease Prc contributes to the pathogen's ability to evade complement-mediated killing in the serum. Here, we further investigated the role of the Prc protease in ExPEC-induced UTIs and the underlying mechanism. METHODS: The uropathogenic role of Prc was determined in a mouse model of UTIs. Using global quantitative proteomic analyses, we revealed that the expression of FliC and other outer membrane-associated proteins was altered by Prc deficiency. Comparative transcriptome analyses identified that Prc deficiency affected expression of the flagellar regulon and genes that are regulated by five extracytoplasmic signaling systems. RESULTS: A mutant ExPEC with a prc deletion was attenuated in bladder and kidney colonization. Global quantitative proteomic analyses of the prc mutant and wild-type ExPEC strains revealed significantly reduced flagellum expression in the absence of Prc, consequently impairing bacterial motility. The prc deletion triggered downregulation of the flhDC operon encoding the master transcriptional regulator of flagellum biogenesis. Overexpressing flhDC restored the prc mutant's motility and ability to colonize the UT, suggesting that the impaired motility is responsible for attenuated UT colonization of the mutant. Further comparative transcriptome analyses revealed that Prc deficiency activated the σE and RcsCDB signaling pathways. These pathways were responsible for the diminished flhDC expression. Finally, the activation of the RcsCDB system was attributed to the intracellular accumulation of a known Prc substrate Spr in the prc mutant. Spr is a peptidoglycan hydrolase and its accumulation destabilizes the bacterial envelope. CONCLUSIONS: We demonstrated for the first time that Prc is essential for full ExPEC virulence in UTIs. Our results collectively support the idea that Prc is essential for bacterial envelope integrity, thus explaining how Prc deficiency results in an attenuated ExPEC.

Peptidoglycan Endopeptidase Spr of Uropathogenic Escherichia coli Contributes to Kidney Infections and Competitive Fitness During Bladder Colonization.

Uropathogenic E scherichia coli (UPEC) is the most common pathogen of urinary tract infections (UTIs). Antibiotic therapy is the conventional measure to manage such infections. However, the rapid emergence of antibiotic resistance has reduced the efficacy of antibiotic treatment. Given that the bacterial factors required for the full virulence of the pathogens are potential therapeutic targets, identifying such factors may facilitate the development of novel therapeutic strategies against UPEC UTIs. The peptidoglycan (PG) endopeptidase Spr (also named MepS) is required for PG biogenesis in E. coli. In the present study, we found that Spr deficiency attenuated the ability of UPEC to infect kidneys and induced a fitness defect during bladder colonization in a mouse model of UTI. Based on the liquid chromatography (LC)/mass spectrometry (MS)/MS analysis of the bacterial envelope, spr deletion changed the levels of some envelope-associated proteins, suggesting that Spr deficiency interfere with the components of the bacterial structure. Among the proteins, FliC was significantly downregulated in the spr mutant, which is resulted in reduced motility. Lack of Spr might hinder the function of the flagellar transcriptional factor FlhDC to decrease FliC expression. The motility downregulation contributed to the reduced fitness in urinary tract colonization. Additionally, spr deletion compromised the ability of UPEC to evade complement-mediated attack and to resist intracellular killing of phagocytes, consequently decreasing UPEC bloodstream survival. Spr deficiency also interfered with the UPEC morphological switch from bacillary to filamentous shapes during UTI. It is known that bacterial filamentation protects UPEC from phagocytosis by phagocytes. In conclusion, Spr deficiency was shown to compromise multiple virulence properties of UPEC, leading to attenuation of the pathogen in urinary tract colonization and bloodstream survival. These findings indicate that Spr is a potential antimicrobial target for further studies attempting to develop novel strategies in managing UPEC UTIs.

Putative Probiotic Strains Isolated from Kefir Improve Gastrointestinal Health Parameters in Adults: a Randomized, Single-Blind, Placebo-Controlled Study.

The dairy products remain as the largest reservoir for isolation of probiotic microorganisms. While probiotics have been immensely reported to exert various health benefits, it is also a common notion that these health potentials are strain and host dependent, leading to the need of more human evidence based on specific strains, health targets, and populations. This randomized, single-blind, and placebo-controlled human study aimed to evaluate the potential benefits of putative probiotic strains isolated from kefir on gastrointestinal parameters in fifty-six healthy adults. The consumption of AB-kefir (Bifidobacterium longum, Lactobacillus acidophilus, L. fermentum, L. helveticus, L. paracasei, L. rhamnosus, and Streptococcus thermophiles; total 10 log CFU/sachet) daily for 3 week reduced symptoms of abdominal pain, bloating (P = 0.014), and appetite (P = 0.041) in male subjects as compared to the control. Gut microbiota distribution profiles were shifted upon consumption of AB-kefir compared to baseline, where the abundance of bifidobacteria was increased in male subjects and maintained upon cessation of AB-kefir consumption. The consumption of AB-kefir also increased gastrointestinal abundance of total anaerobes (P = 0.038) and total bacterial (P = 0.049) in female subjects compared to the control after 3 weeks. Our results indicated that AB-kefir could potentially be developed as a natural strategy to improve gastrointestinal functions in adults.

Group A Streptococcus Induces LAPosomes via SLO/ß1 Integrin/NOX2/ROS Pathway in Endothelial Cells That Are Ineffective in Bacterial Killing and Suppress Xenophagy.

Group A streptococcus (GAS) is an important human pathogen which can cause fatal diseases after invasion into the bloodstream. Although antibiotics and immune surveillance are the main defenses against GAS infection, GAS utilizes internalization into cells as a major immune evasion strategy. Our previous findings revealed that light chain 3 (LC3)-associated single membrane GAS-containing vacuoles in endothelial cells are compromised for bacterial clearance due to insufficient acidification after fusion with lysosomes. However, the characteristics and the activation mechanisms of these LC3-positive compartments are still largely unknown. In the present study, we demonstrated that the LC3-positive GAS is surrounded by single membrane and colocalizes with NADPH oxidase 2 (NOX2) complex but without ULK1, which are characteristics of LC3-associated phagocytosis (LAP). Inhibition of NOX2 or reactive oxygen species (ROS) significantly reduces GAS multiplication and enhances autolysosome acidification in endothelial cells through converting LAP to conventional xenophagy, which is revealed by enhancement of ULK1 recruitment, attenuation of p70s6k phosphorylation, and formation of the isolation membrane. We also clarify that the inactivation of mTORC1, which is the initiation signal of autophagy, is inhibited by NOX2- and ROS-activated phosphatidylinositol 3-kinase (PI3K)/AKT and MEK/extracellular signal-regulated kinase (ERK) pathways. In addition, streptolysin O (SLO) of GAS is identified as a crucial inducer of ROS for ß1 integrin-mediated LAP induction. After downregulation of ß1 integrin, GAS multiplication is reduced, accompanied with LAP inhibition and xenophagy induction. These results demonstrate that GAS infection preferentially induces ineffective LAP to evade xenophagic killing in endothelial cells through the SLO/ß1 integrin/NOX2/ROS pathway.IMPORTANCE Our previous reports showed that the LC3-associated GAS-containing single membrane vacuoles are inefficient for bacterial clearance in endothelial cells, which may result in bacteremia. However, the characteristics and the induction mechanisms of these LC3-positive vacuoles are still largely unknown. Here we provide the first evidence that these LC3-positive GAS-containing single membrane compartments appear to be LAPosomes, which are induced by NOX2 and ROS. Through NOX2- and ROS-mediated signaling, GAS preferentially induces LAP and inhibits bacteriostatic xenophagy in endothelial cells. We also provide the first demonstration that ß1 integrin acts as the receptor for LAP induction through GAS-produced SLO stimulation in endothelial cells. Our findings reveal the underlying mechanisms of LAP induction and autophagy evasion for GAS multiplication in endothelial cells.

HECT E3 Ubiquitin Ligase-Regulated Txnip Degradation Facilitates TLR2-Mediated Inflammation During Group A Streptococcal Infection.

Thioredoxin-interacting protein (Txnip) inhibits the activity of thioredoxin (Trx) to modulate inflammatory responses. The burden of inflammation caused by microbial infection is strongly associated with disease severity; however, the role of Txnip in bacterial infection remains unclear. In Group A Streptococcus (GAS)-infected macrophages, Txnip was degraded independent of glucose consumption and streptococcal cysteine protease expression. Treatment with proteasome inhibitors reversed GAS-induced Txnip degradation. The activation of Toll-like receptor 2 (TLR2) initiated Txnip degradation, while no further Txnip degradation was observed in TLR2-deficient bone marrow-derived macrophages. NADPH oxidase-regulated NF-κB activation and pro-inflammatory activation were induced and accompanied by Txnip degradation during GAS infection. Silencing Txnip prompted TLR2-mediated inducible nitric oxide synthase (iNOS)/NO, TNF-α, and IL-6 production whereas the blockage of Txnip degradation by pharmacologically inhibiting the HECT E3 ubiquitin ligase with heclin and AMP-dependent protein kinase with dorsomorphin effectively reduced such effects. Our findings reveal that TLR2/NADPH oxidase-mediated Txnip proteasomal degradation facilitates pro-inflammatory cytokine production during GAS infection.

Susceptibility rates of clinically important bacteria collected from intensive care units against colistin, carbapenems, and other comparative agents: results from Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART).

OBJECTIVES: This study aimed to determine the in vitro susceptibility of commonly encountered Gram-negative bacilli (GNB) recovered from patients admitted to intensive care units (ICUs) in Taiwan against colistin, carbapenems, and other comparative agents. METHODS: In total, 758 nonduplicate GNB isolates were obtained from clinical specimens of ICU patients at seven medical centers in 2016. Minimum inhibitory concentrations (MICs) were determined using the Vitek 2 susceptibility system. The reference broth-microdilution method was performed to determine MICs of colistin. Five main carbapenemase genes among carbapenem-non-susceptible GNB and mcr-1-mcr5 genes among colistin non-wild-type or -resistant isolates were determined. RESULTS: After exclusion 38 Proteus mirabilis and 13 Morganella morganii spp. among 361 Enterobacteriaceae isolates, 34 (9.4%) isolates were carbapenem-insusceptible, 91.1% (n=31) were colistin wild type, and three and one Klebsiella pneumoniae isolates carried bla KPC and bla OXA48-like, respectively. Carbapenem-insusceptible isolates were found in 23.4% (30 of 128) and 63.0% (87 of 138) of isolates of the Pseudomonas aeruginosa and Acinetobacter baumannii complex, respectively. mcr-1 was detected in two (1.8%) Enterobacter cloacae isolates. Very major errors between two methods of susceptibility to colistin were found in 1.5% of K. pneumoniae, 27.5% of E. cloacae, 4.7% of P. aeruginosa, and 10.1% of A. baumannii complex isolates. CONCLUSION: In this study, 8.7% of Enterobacteriaceae isolates from ICUs were not susceptible to carbapenem, and bla KPC and bla OXA48-like were found among three and one carbapenem-insusceptible K. pneumoniae isolates, respectively. Colistin MICs determined by Vitek 2 were not reliable, especially for E. cloacae and A. baumannii complex isolates.

Group A Streptococcus Subcutaneous Infection-Induced Central Nervous System Inflammation Is Attenuated by Blocking Peripheral TNF.

Group A streptococcus (GAS) infection causes a strong inflammatory response associated with cytokine storms, leading to multiorgan failure, which is characterized as streptococcal toxic shock syndrome. However, little is known about GAS subcutaneous infection-mediated brain inflammation. Therefore, we used a bioluminescent GAS strain and reporter mice carrying firefly luciferase under transcriptional control of the nuclear factor-kappa B (NF-κB) promoter to concurrently monitor the host immune response and bacterial burden in a single mouse. Notably, in addition to the subcutaneous inoculation locus at the back of mice, we detected strong luminescence signals from NF-κB activation and increased inflammatory cytokine production in the brain, implying the existence of central nervous system inflammation after GAS subcutaneous infection. The inflamed brain exhibited an increased expression of glial fibrillary acidic protein and nicotinamide adenine dinucleotide phosphate oxidase components and greater microglial activation and blood-brain barrier (BBB) disruption. Furthermore, Fluoro-Jade C positive cells increased in the brain, indicating that neurons underwent degeneration. Peripheral tumor necrosis factor (TNF), which contributes to pathology in brain injury, was elevated in the circulation, and the expression of its receptor was also increased in the inflamed brain. Blockage of peripheral TNF effectively reduced brain inflammation and injury, thereby preventing BBB disruption and improving survival. Our study provides new insights into GAS-induced central nervous system inflammation, such as encephalopathy, which can be attenuated by circulating TNF blockage.