Antagonistic Activities of Lactobacillus rhamnosus JB3 Against Helicobacter pylori Infection Through Lipid Raft Formation

Helicobacter pylori is a Gram-negative pathogen that can increase the risk of stomach cancer in infected patients. H. pylori exploits lipid rafts to infect host cells. Infection triggers clustering of Lewis x antigen (Lex) and integrins in lipid rafts to facilitate H. pylori adherence to the gastric epithelium. H. pylori infection can be treated with probiotics containing lactic acid bacteria that offer numerous benefits to the host while lacking the side effects associated with antibiotic therapy. Previously, we showed that the cell-free supernatant (CFS) derived from Lactobacillus rhamnosus JB3 (LR-JB3) at a multiplicity of infection (MOI) of 25 attenuated the pathogenicity of H. pylori. In this study, we established a mucin model to simulate the gastric environment and to further understand the influence of mucin on the pathogenesis of H. pylori. Porcine stomach mucin dramatically upregulated H. pylori virulence gene expression, including that of babA, sabA, fucT, vacA, hp0499, cagA, and cagL, as well as the adhesion and invasion ability of H. pylori and induced increased levels of IL-8 in infected-AGS cells. The CFS derived from LR-JB3 at a MOI of 25 reduced the expression of H. pylori sabA, fucT, and hp0499 in mucin, as well as that of the Lex antigen and the α5β1 integrin in AGS cells during co-cultivation. These inhibitory effects of LR-JB3 also suppressed lipid raft clustering and attenuated Lewis antigen-dependent adherence, type IV secretion system-mediated cell contact, and lipid raft-mediated entry of VacA to host cells. In conclusion, LR-JB3 could affect H. pylori infection through mediating lipid raft formation of the host cells. The currently unknown cues secreted from LR-JB3 are valuable not only for treating H. pylori infection, but also for treating diseases that are also mediated by lipid raft signaling, such as cancer and aging-associated and neurodegenerative conditions.

VicRK and CovR polymorphisms in Streptococcus mutans strains associated with cardiovascular infections

Introduction. Streptococcus mutans , a common species of the oral microbiome, expresses virulence genes promoting cariogenic dental biofilms, persistence in the bloodstream and cardiovascular infections. Gap statement. Virulence gene expression is variable among S. mutans strains and controlled by the transcription regulatory systems VicRK and CovR. Aim. This study investigates polymorphisms in the vicRK and covR loci in S. mutans strains isolated from the oral cavity or from the bloodstream, which were shown to differ in expression of covR, vicRK and downstream genes. Methodology. The transcriptional activities of covR, vicR and vicK were compared by RT-qPCR between blood and oral strains after exposure to human serum. PCR-amplified promoter and/or coding regions of covR and vicRK of 18 strains (11 oral and 7 blood) were sequenced and compared to the reference strain UA159. Results. Serum exposure significantly reduced covR and vicR/K transcript levels in most strains (P<0.05), but reductions were higher in oral than in blood strains. Single-nucleotide polymorphisms (SNPs) were detected in covR regulatory and coding regions, but SNPs affecting the CovR effector domain were only present in two blood strains. Although vicR was highly conserved, vicK showed several SNPs, and SNPs affecting VicK regions important for autokinase activity were found in three blood strains. Conclusions. This study reveals transcriptional and structural diversity in covR and vicR/K, and identifies polymorphisms of functional relevance in blood strains, indicating that covR and vicRK might be important loci for S. mutans adaptation to host selective pressures associated with virulence diversity.

Effect of Enterocins A and B on the Viability and Virulence Gene Expression of Listeria monocytogenes in Sliced Dry-Cured Ham

Dry-cured ham can be contaminated with Listeria monocytogenes during its industrial processing. The use of bacteriocins could ensure the safety of such meat products, but their effect on pathogen physiology is unknown. Therefore, the impact of enterocins A and B on the L. monocytogenes population, and the expression patterns of five genes (inlA, inlB, clpC, fbpA and prfA) related to adhesion/invasion and virulence regulation have been monitored in sliced dry-cured ham during 30 d of storage in refrigeration (4 °C) and temperature-abuse conditions (20 °C). L. monocytogenes strains S2 (serotype 1/2a) and S7-2 (serotype 4b) counts were reduced by 0.5 and 0.6 log units immediately after the application of enterocins A and B, a decrease lower than previously reported. Differences in gene expression were found between the two strains. For strain S2, expression tended to increase for almost all genes up to day seven of storage, whereas this increase was observed immediately after application for strain S7-2; however, overall gene expression was repressed from day one onwards, mainly under temperature-abuse conditions. L. monocytogenes strains investigated in the present work exhibited a mild sensitivity to enterocins A and B in sliced dry-cured ham. Bacteriocins caused changes in the expression patterns of virulence genes associated with adhesion and invasion, although the potential virulence of surviving cells was not enhanced.

Evaluation of antimicrobial effects of photo-sonodynamic antimicrobial chemotherapy based on nano-micelle curcumin on virulence gene expression patterns in Acinetobacter baumannii

Background: Abaumannii baumannii rapidly resistance to a wide range of antimicrobial agents. The combination of antimicrobial photodynamic therapy (aPDT) and sonodynamic antimicrobial chemotherapy (SACT) known as photo-sonodynamic antimicrobial chemotherapy (PSACT) has received considerable attention as one of the emerging and promising strategies against microbial infections. Objective: This study aimed to investigate the antimicrobial effects of PSACT based on nano-micelle curcumin (N-MCur) on the virulence gene expression patterns in A. baumannii. Materials and methods: N-MCur as a photo-sonosensitizer was synthesized and confirmed. To determine sub-significant reduction dose of PSACT, sub-significant reduction dose of N-MCur and blue laser light during aPDT, and ultrasound power output during SACT were assessed. Finally, changes in the expression of genes involved in treated A. baumannii by minimum sub-significant reduction dose of PSACT were determined using quantitative real-time-PCR (qRT-PCR). Results: PSACT using 12.5 mM N-MCur at the ultrasound power outputs of 28.7, 36.9, and 45.2 mW/cm2 with 4 min irradiation time of blue laser, as well as, 6.2 mM N-MCur at an ultrasound power output of 45.2 mW/cm2 plus 3 min blue laser irradiation time exhibited the significant dose-dependent reduction against A. baumannii cell viability compared to the control group (P<0.05). After treatment of A. baumannii using 3.1 mM N-MCur + 2 min blue laser irradiation time + 28.7 mW/cm2 ultrasound as the minimum sub-significant reduction doses of PSACT, mRNA expression was significantly upregulated to 6.0-, 11.2-, and 13.7-folds in recA, blsA, and dnaK and downregulated to 8.6-, 10.1-, and 14.5-folds in csuE, espA, and abaI, respectively. Conclusions: N-MCur-mediated PSACT could regulate the expression of genes involved in A. baumannii pathogenesis. Therefore, PSACT can be proposed as a promising application to treat infections caused by A. baumannii.

Preliminary study into the effects of tobacco smoke on Candida albicans

Background: Denture-stomatitis (DS) is the most common form of oral candidosis with increased prevalence in cigarette smokers (Akram et al. 2018). Interestingly, tobacco condensate (TC) increases Candida albicans adhesion, growth, biofilm-formation, virulence gene expression (Semlali et al. 2014)and hyphal production (Awad and Karuppayil 2018). We hypothesised that TC-treated denture acrylic would therefore affect C. albicans within acrylic biofilms. Methods: Acrylic discs (pre-conditioned with TC, artificial saliva (AS) or water) were incubated at 37°C with C. albicans (n=6) for 90 min or 24 h. Adherent Candida were stained with calcofluor white and confocal laser scanning microscopy (CLSM) used to assess levels of adherence, biofilm and hyphal numbers. Expressed virulence genes (n=7) were measured by qPCR. Results: CLSM showed that effects of TC-treatment were strain dependent. Adherence of C. albicans PTR/94 to TC-treated surfaces was significantly (P<0.002) lower than on the untreated control. Biofilm levels of PTR/94 after 24 h were found to be significantly higher on AS-treated acrylic than the TC-treated and untreated control. Five strains had significantly fewer filamentous forms after 90 min on TC-treated surfaces. TC-treatment promoted hyphal levels for strain 705/93 after 24h. Conclusion: TC pre-conditioning altered adherence and biofilm coverage of C. albicans to acrylic surfaces and influenced hyphal development. Work is ongoing to ascertain the significance of these effects on C. albicans pathogenicity. Akram et al. (2018). Journal of Oral Science 60(1):115–120. Awad and Karuppayil (2018). American Journal of Clinical Microbiology and Antimicrobials1(3):1–6. Semlali et al. (2014). BMC Microbiology. 14:61

BrnQ-type Branched-chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence

Bacillus anthracis, the anthrax agent, exhibits robust proliferation in diverse niches of mammalian hosts. Metabolic attributes of B. anthracis that permit rapid growth in multiple mammalian tissues have not been established. We posit that branched-chain amino acid (BCAA: Isoleucine, leucine and valine) metabolism is key to B. anthracis pathogenesis. Increasing evidence indicates relationships between B. anthracis virulence and expression of BCAA-related genes. Expression of some BCAA-related genes is altered during culture in bovine blood in vitro and the bacterium exhibits valine auxotrophy in a blood serum mimic medium. Transcriptome analyses have revealed that the virulence regulator AtxA, that positively affects expression of the anthrax toxin and capsule genes, negatively regulates genes predicted to be associated with BCAA biosynthesis and transport. Here, we show that B. anthracis growth in defined media is severely restricted in the absence of exogenous BCAAs, indicating that BCAA transport is required for optimal growth in vitro. We demonstrate functional redundancy among multiple BrnQ-type BCAA transporters. Three transporters are associated with isoleucine and valine transport, and deletion of one, BrnQ3, attenuates virulence in a murine model for anthrax. Interestingly, an ilvD-null mutant lacking dihydroxy-acid dehydratase, an enzyme essential for BCAAs synthesis, exhibits unperturbed growth when cultured in media containing BCAAs, but is highly attenuated in the murine model. Finally, our data show that BCAAs enhance AtxA activity in a dose-dependent manner, suggesting a model in which BCAAs serve as a signal for virulence gene expression.

Molecular Characteristics of Streptococcus pyogenes Isolated From Chinese Children With Different Diseases

Streptococcus pyogenes is a bacterial pathogen that causes a wide spectrum of clinical diseases exclusively in humans. The distribution of emm type, antibiotic resistance and virulence gene expression for S. pyogenes varies temporally and geographically, resulting in distinct disease spectra. In this study, we analyzed antibiotic resistance and resistance gene expression patterns among S. pyogenes isolates from pediatric patients in China and investigated the relationship between virulence gene expression, emm type, and disease categories. Forty-two representative emm1.0 and emm12.0 strains (n = 20 and n = 22, respectively) isolated from patients with scarlet fever or obstructive sleep apnea-hypopnea syndrome were subjected to whole-genome sequencing and phylogenetic analysis. These strains were further analyzed for susceptibility to vancomycin. We found a high rate and degree of resistance to macrolides and tetracycline in these strains, which mainly expressed ermB and tetM. The disease category correlated with emm type but not superantigens. The distribution of vanuG and virulence genes were associated with emm type. Previously reported important prophages, such as φHKU16.vir, φHKU488.vir, Φ5005.1, Φ5005.2, and Φ5005.3 encoding streptococcal toxin, and integrative conjugative elements (ICEs) such as ICE-emm12 and ICE-HKU397 encoding macrolide and tetracycline resistance were found present amongst emm1 or emm12 clones from Shenzhen, China.

Quorum Sensing Regulation of Virulence Gene Expression in Vibrio harveyi during its Interaction with Marine Diatom Skeletonema marinoi

Communication between species from different kingdoms may be as important as intra-kingdom communication. It has recently been confirmed that co-existing bacteria and phytoplankton in aquatic ecosystems do cross-talk. This study examined the signs of possible cross signalling between V. harveyi, one of the predominant bacterial species of the marine ecosystem and a dominant diatom species, S.marinoi, to understand communication over species borders. It is known that V.harveyi employ quorum sensing for cell-to-cell communication, bioluminescence (luxR), and the regulation of the virulence gene (vhp, chiA). Former studies have also shown, this kind of interactions being disrupted by compounds secreted by a few algal species existing in the aquatic ecosystem. We investigated the QS communication by quantifying the expression levels of virulence regulator luxR and virulence factors metalloprotease (vhp) and chitinase (chiA) in four different V. harveyi strains grown in the presence of S. marinoi strain. Results obtained in this study indicate that quorum sensing was activated in strains of V. harveyi analysed but did not regulate the expressions of vhp and chiA virulence factors. This observation suggests that the existence of S. marinoi did not interfere with the QS behaviour of V. harveyi and its interaction with marine diatom; it may be due to the commensalism relationship.

1006. Association of Development of Pneumonia and Virulence Gene Expression in Acinetobacter baumannii Isolated from Clinical Specimens

Background Not all Acinetobacter baumannii isolated from respiratory specimens are true pathogens. Distinguishing between true pathogens and colonizers is important to initiate early treatment and to reduce the unnecessary prescription of antibiotics. To determine the microbiological factors contributing to the development of A. baumannii pneumonia, we investigated the association between the expression level of known A. baumannii virulence genes such as ompA and hisF and pneumonia. Methods Patients in whose respiratory specimens A. baumannii was identified between January 2018 and January 2019 in a tertiary university hospital were recruited into this study. Relevant radiologic findings and more than 5 days of susceptible antibiotic prescription started within 3 days of bacterial isolation were considered as having pneumonia. The absence of radiologic findings of pneumonia until 7 days after the isolation of A. baumannii was defined as colonization. The expression of ompA and hisF was determined with quantitative reverse-transcription polymerase chain reaction. Host factors known to be associated with pneumonia and expression levels of virulent genes were compared between the groups. Results Overall, 246 patients in whose respiratory specimens A. baumannii was identified were recruited into this study. Among them, 17 and 24 patients were assigned to the pneumonia and colonizer groups, respectively. In the univariable analysis, ompA, ICU stay, and mechanical ventilation were significantly associated with pneumonia (p = 0.03, &lt; 0.01, &lt; 0.01 respectively). In the multivariable analysis, mechanical ventilation was significantly associated with pneumonia (OR = 9.75, p = 0.03). ompA expression was not significantly associated with pneumonia in the multivariable analysis (OR = 1.12, p = 0.75) (Table 1). ompA and hisF were significantly associated with the 30-day in-hospital mortality (p = 0.02, &lt; 0.01). Table 1. Univariable and multivariable analysis of factors related to pneumonia Conclusion The association between increased ompA expression in A. baumannii and the development of pneumonia was not statistically significant after adjusting for patient factors. However, the relatively high expression of ompA in pneumonia patients and their association with increased mortality suggests the need for larger-scale prospective studies to draw a conclusion. Disclosures All Authors: No reported disclosures

An In-Vitro Study of Molecular Effects of a Combination Treat-ment with Antibiotics and Nanofluid Containing Carbon Nano-tubes on Klebsiella pneumoniae

Background: We aimed to prepare a nanofluid, containing f-MWCNTs, and investigate the antibacterial efficacy of f-MWCNTs+ ciprofloxacin (cip) on Klebsiella pneumoniae by evaluating the virulence gene expression. Methods: This study was carried out from 2019 to 2020, in the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran. The nanofluid containing antibiotic and f-MWCNTs were prepared by the ultrasonic method. The minimum inhibitory concentrations (MICs) of ciprofloxacin and f-MWCNTs were determined using the broth micro dilution MIC tests. For examining the antibacterial effects, the expression level of virulence genes, under the influence of f-MWCNTs, was evaluated by a real-time PCR. Results: The effect of 8 µg/ml ciprofloxacin + 400 µg/ml f-MWCNTs, completely inhibited the growth of the resistant isolate of K. pneumoniae, while, in the ATCC 700,603 isolate, 2 µg/ml ciprofloxacin with 100 µg/ml f-MWCNT could inhibit a bacterial growth. In the resistant K. pneumoniae clinical isolate, after f-MWCNT+cip treatment, the expression of fimA, fimD, wza, and wzi genes was significantly downregulated, compared to the ciprofloxacin treatment, and upregulated, compared to the negative control. For the ATCC 700,603 isolate treated with f-MWCNT+cip, the expression of fimA, fimD and wza virulence genes showed upregulation, compared to the negative control and downregulated in comparison with the ciprofloxacin treatment. Conclusion: Simultaneous treatment of resistant isolate of K. pneumoniae with f-MWCNTs +antibiotic could improve the effectiveness of antibiotic at lower doses, due to the reduced expression of virulence genes in comparison with antibiotic treatment, besides the increased cell wall permeability to antibiotics.