Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Historical and Molecular Perspectives on the Presence of Helicobacter pylori in Latin America: A Niche to Improve Gastric Cancer Risk Assessment.

Helicobacter pylori (H. pylori) is responsible for causing chronic gastritis, which can cause peptic ulcer and premalignant lesions such as atrophic gastritis, intestinal metaplasia, and dysplasia, with the risk of developing gastric cancer. Recent data describe that H. pylori colonizes the gastric mucosa of more than 50% of the world's population; however, this bacterium has been described as infecting the human population since its prehistory. This review focuses on the populations and subpopulations of H. pylori, differentiated by the polymorphisms present in their constitutive and virulence genes. These genes have spread and associated with different human populations, showing variability depending on their geographical distribution, and have evolved together with the human being. The predominant genotypes worldwide, Latin America and Chile, are described to understand the genetic diversity and pathogenicity of H. pylori in different populations and geographic regions. The high similarity in the sequence of virulence genes between H. pylori strains present in Peruvian and Spanish natives in Latin America suggests a European influence. The presence of cagA-positive strains and vacA s1 m1 allelic variants is observed with greater prevalence in Chilean patients with more severe gastrointestinal diseases and is associated with its geographical distribution. These findings highlight the importance of understanding the genetic diversity of H. pylori in different regions of the world for a more accurate assessment of the risk of associated diseases and their potential impact on health.

Assessment of the mixed origin of the gastric epithelial extracellular vesicles in acellular transfer of Helicobacter pylori toxins and a systematic review.

BACKGROUND: H. pylori are generally considered as extracellular organisms, with exclusive colonization of the gastric milieu. Yet, several extra gastric manifestations are associated with this infection. The aim of the present study was to investigate the feasibility of toxin transfer by extracellular vesicles, from bacterial and epithelial origins. METHODS: Tox-positive H. pylori and its two cagA and vacA mutant strains were used to produce bacterial vesicles (BVs) and to infect AGS cells. The produced BVs and the infected cell vesicles (ICVs) were collected by ultracentrifugation and evaluated by western blotting, DLS and electron microscopy. These two sets of vesicles were applied to a second set of recipient AGS cells, in which the acellular transfer of toxins, IL-8 production and downstream morphologic changes were assessed, by western blotting, ELISA and light microscopy, respectively. RESULTS: The BVs were positive for H. pylori membrane markers (BabA and UreB), VacA and CagA toxins, except for from the corresponding mutant strains. The ICVs were larger in size and positive for bacterial markers, as well as epithelial markers of CD9, LGR5, but negative for nuclear (Ki76) or cytoplasmic (ß-actin) markers. Bacteria-independent transfer of CagA and VacA into the recipient cells occurred upon treatment of cells with BVs and ICVs, followed by cellular vacuolation and elongation. IL-8 production was induced in recipient AGS cells, treated with BVs (1279.4 ± 19.79 pg/106 cells), early (8 h, 1171.4 ± 11.31 pg/106 cells) and late (48 h, 965.4 ± 36.77 pg/106 cells) ICVs (P < 0.0001). CONCLUSION: Our data indicates that ICVs, with mixed bacterial and epithelial constituents, similar to BVs, are capable of transferring bacterial toxins into the recipient cells, inducing IL-8 production and subsequent morphologic changes, in an acellular manner.

Assessment of recombinant antigens Tp0100 and Tp1016 of Treponema pallidum for serological diagnosis of syphilis.

OBJECTIVE: To discover novel serodiagnostic candidates for the serological diagnosis of syphilis. METHODS: Two recombinant Treponema pallidum proteins Tp0100 and Tp1016 were expressed, purified, and identified by Western Blotting. A total of 600 clinical serum samples were tested with the Tp0100-based ELISA, the Tp1016-based ELISA, and the commercial LICA Syphilis TP kit (ChIVD, Beijing, China). The sensitivities were determined by testing 340 samples from individuals with clinically diagnosed primary, secondary, latent, and tertiary syphilis. The specificities were determined by screening 260 samples from healthy controls and individuals with potentially cross-reactive infections, including leptospirosis, Lyme disease, hepatitis B, tuberculosis, rheumatoid arthritis, systemic lupus erythematosus. Kappa (κ) values were applied to compare the agreement between clinical syphilis diagnosis and the Tp0100-based ELISA, the Tp1016-based ELISA, or the LICA Syphilis TP test. RESULTS: Using clinical syphilis diagnosis as the gold standard, Tp0100 exhibited an overall sensitivity of 95.6% and specificity of 98.1% for testing IgG antibody while Tp1016 demonstrated only an overall sensitivity of 75.0% and specificity of 79.6%. In contrast, the LICA Syphilis TP test revealed an overall sensitivity of 97.6% and specificity of 96.2%. In addition, the overall percent agreement and corresponding κ values were 96.7% (95% CI 95.6%-97.8%) and 0.93 for the Tp0100-based ELISA, 77.0% (95% CI 74.3%-79.7%) and 0.54 for the Tp1016-based ELISA, and 97.0% (95% CI 96.0%-98.0%) and 0.94 for the LICA Syphilis TP test, respectively. CONCLUSION: The recombinant T. pallidum protein Tp0100 shows promise as a novel diagnostic antigen in the serological tests for syphilis.

Risk assessment of gastric cancer in the presence of Helicobacter pylori cagA and hopQII genes.

Helicobacter pylori bacterium is one of the most common bacterial infections globally and is the leading cause of indigestion, gastric and duodenal ulcers, and gastric cancer. This bacterium can escape the antibacterial effects of stomach acid by adapting to the inner layers of the stomach. It combines with the natural sugars in the gastric mucosa. The compound is so effective that it makes bacterium resistant. For genes related to the pathogenesis of H. pylori, using the existence of genes such as cagA, hopQI, and hopQII, PCR is performed on some of these genes to amplify fragments of different lengths. One of the less-studied cases is that two or more pathogenic genes are simultaneously associated with H. pylori. This study examined the frequency of diseases and healthy individuals infected with H. pylori and cagA and hopQII genotypes. To diagnose H. pylori infection in healthy and stomach cancer patients, the PCR products are electrophoresed on the agarose gel after glmM gene amplification by PCR. To this aim, stomach tissue biopsies were used for patients, and saliva was used for healthy individuals. For this purpose, 150 gastric biopsy samples from stomach cancer patients and 150 saliva samples from healthy people were collected. Data showed a significant relationship between the coexistence of two genes, cagA and hopQII, and stomach cancer. 34.2% of patients and 10.1% of healthy individuals showed two genotypes, while other healthy people (89.9%) infected with H. pylori did not have this genotype. Therefore, the simultaneous presence of these two bacterial genes in human societies can be an essential biomarker for the diagnosis and prognosis of gastric cancer.

Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis.

The diagnosis of abdominal tuberculosis (aTB) is challenging and there is an urgent need for an accurate diagnostic test. We have developed a high affinity DNA aptamer against GlcB antigen of Mycobacterium tuberculosis (Mtb). We further compared the diagnostic utility of in-house-generated high affinity DNA aptamers and polyclonal antibodies against two Mtb antigens, namely GlcB and HspX, in ascitic fluid samples. These diagnostic reagents were assessed in patients (n = 94) who were categorized as 'Definite TB', 'Probable TB', 'Possible TB' (taken together as aTB) and 'Non-TB' disease. Receiver operating characteristic curves were used to derive cut-off values to provide ≥93% specificity. Aptamer Linked Immobilized Sorbent Assay (ALISA) for HspX and GlcB exhibited a sensitivity of ∼84% and 50%, respectively (p-value <0.01). In contrast, antibody-based ELISA exhibited a lower sensitivity of ∼18% and ∼28% for HspX and GlcB, respectively (p-value <0.0001 and p = 0.05 for HspX and GlcB ELISA vs. ALISA, respectively). HspX ALISA detected 32/38 aTB cases, while Xpert detected only 9 samples. In conclusion, HspX aptamer-based test was found to be superior to the other tests for diagnosing aTB and it nearly fulfils the sensitivity criteria of WHO's 'Target Product Profile' for extrapulmonary tuberculosis (sensitivity ≥80%, specificity 98%).

Comparative assessment of the results of detection of СagА-positive strains of Helicobacter pylori by molecular-genetic and immunochromatographic methods in different biological materials.

A comparative analysis of the detection of CagA-positive strains of H. pylori by immunochromatographic and molecular genetic methods was carried out. We used H. pylori strains isolated from individuals with diseases of the gastrointestinal tract. The immunochromatographic method was implemented using a developed experimental model of an immunochromatographic test system for detecting the H. pylori CagA protein in various biological materials. Determination of the pathogenicity gene cagA of H. pylori was carried out using the «Helikopol SA¼ test system («Litekh¼, Russia). The assessment of the comparability of the results of detecting CagA-positive strains of H. pylori was carried out using statistical methods: Monte-Carlo, calculation of the chi-square test (χ2) and Kendall's τ-b and Somer's d coefficients. Statistical analysis was performed using the software packages «Microsoft Office Excel¼, «Statistica 10.0¼, «WinBUGS 1.4.0.¼ The study showed the absence of a statistically significant difference and the presence of a direct strong correlation between the results of detecting CagA-positive strains by molecular genetic and immunochromatographic methods, which indicates that these methods provide similar results in identifying highly pathogenic strains of H. pylori.

Methods for Assessment of OMV/GMMA Quality and Stability.

Outer membrane vesicles (OMV) represent a promising platform for the development of vaccines against bacterial pathogens. More recently, bacteria have been genetically modified to increase OMV yield and modulate the design of resulting particles, also named generalized modules for membrane antigens (GMMA). OMV/GMMA resemble the bacterial surface of the pathogen, where key antigens to elicit a protective immune response are and contain pathogen-associated molecular patterns (e.g., lipopolysaccharides, lipoproteins) conferring self-adjuvanticity. On the other hand, OMV/GMMA are quite complex molecules and a comprehensive panel of analytical methods is needed to ensure quality, consistency of manufacture and to follow their stability over time. Here, we describe several procedures that can be used for OMV/GMMA characterization as particles and for analysis of key antigens displayed on their surface.

Rapid Detection of Carbapenemase-producing Enterobacteriaceae From Blood Culture Bottles of Known CPE Carriers: Real-world Experience.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy.

Assessment and utility of 2 Chlamydia trachomatis Pgp3 serological assays for seroprevalence studies among women in the United States.

Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013-2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6-31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5-30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18-39 years old); of these, 42 (2.4%, 95% CI 1.8-3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8-89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8-94.1) were Pgp3-MBA-positive. Although Pgp3-ELISA and Pgp3-MBA demonstrated equivalent performance in this study, an advantage of the Pgp3-MBA over Pgp3-ELISA is that it is well suited for high sample throughput applications.

Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores.

CONTEXT: Bacillus anthracis secretes a tripartite toxin comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). The human anthrax vaccine is mainly composed of the anthrax protective antigen (PA). Considerable efforts are being directed towards improving the efficacy of vaccines because the use of commercial anthrax vaccines (human/veterinary) is associated with several limitations. OBJECTIVE: In this study, a triple chimeric antigen referred to as ELP (gene accession no: MT590758) comprising highly immunogenic domains of PA, LF, and EF was designed, constructed, and assessed for the immunization capacity against anthrax in a guinea pig model. MATERIALS AND METHODS: Immunization was carried out considering antigen titration and immunization protocol. The immunoprotective efficacy of the ELP was evaluated in guinea pigs and compared with the potency of veterinary anthrax vaccine using a challenge test with B. anthracis 17JB strain spores. RESULTS: The results demonstrated that the ELP antigen induced strong humoral responses. The T-cell response of the ELP was found to be similar to PA, and showed that the ELP could protect 100%, 100%, 100%, 80% and 60% of the animals from 50, 70, 90, 100 and 120 times the minimum lethal dose (MLD, equal 5 × 105 spore/ml), respectively, which killed control animals within 48 h. DISCUSSION AND CONCLUSIONS: It is concluded that the ELP antigen has the necessary requirement for proper immunization against anthrax and it can be used to develop an effective recombinant vaccine candidate against anthrax.

emPAI-assisted strategy enhances screening and assessment of Mycobacterium tuberculosis infection serological markers.

Discovering new serological markers of Mycobacterium tuberculosis (MTB) infection and establishing a rapid and efficient detection technology is of great significance for the prevention and control of tuberculosis. In this study, we established an exponentially modified protein abundance index (emPAI) value-assisted strategy to investigate and improve the screening efficiency of serological biomarkers of tuberculosis. First, we used LC-MS/MS to analyse MTB culture filtrate proteins (MTB-CFPs), and 632 MTB proteins were identified. Then, the characteristic values of MTB-CFPs - including emPAI value, molecular weight (Mw), isoelectric point (pI), grand average of hydropathy (GRAVY), transmembrane domain (TMD) and functional groups were calculated. Next, we successfully prepared 10 MTB proteins with emPAI value > 1.0 and recombinantly expressed these proteins in Escherichia coli. At the same time, 3 MTB proteins with emPAI between 0.1 and 0.5 were randomly selected as the control groups, and the immunogenicity of the recombinant MTB proteins was detected using ELISA. The sensitivity and receiver operating characteristic (ROC) curves were calculated for each recombinant MTB protein. The results showed that the areas under the curve (AUC) value of Rv2031c, Rv0577, Rv0831c, Rv0934 and Rv3248c were all higher than those of Rv3875 (AUC, 0.6643). Further analysis of the relationship between emPAI value and antibody sensitivity, AUC value and antibody affinity in mice immunized with recombinant MTB protein showed that emPAI values were positively correlated with them, and R-squared value ranged from 0.64 to 0.79. The only exception was ESAT-6 (encoded by the Rv3875 gene), which AUC value was relatively low owing to its strong immunosuppressive properties. This study provides a rationale for the serological marker screening of emPAI-assisted tuberculosis clinical test. The results also provide new technical support for the screening of candidate serological markers of infectious diseases in the future.

Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines.

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52-90 µg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection.

Bacterial biofilm-derived antigens: a new strategy for vaccine development against infectious diseases.

INTRODUCTION: Microorganisms can develop into a social organization known as biofilms and these communities can be found in virtually all types of environment on earth. In biofilms, cells grow as multicellular communities held together by a self-produced extracellular matrix. Living within a biofilm allows for the emergence of specific properties for these cells that their planktonic counterparts do not have. Furthermore, biofilms are the cause of several infectious diseases and are frequently inhabited by multi-species. These interactions between microbial species are often critical for the biofilm process. Despite the importance of biofilms in disease, vaccine antigens are typically prepared from bacteria grown as planktonic cells under laboratory conditions. Vaccines based on planktonic bacteria may not provide optimal protection against biofilm-driven infections. AREAS COVERED: In this review, we will present an overview of biofilm formation, what controls this mode of growth, and recent vaccine development targeting biofilms. EXPERT OPINION: Previous and ongoing research provides evidence that vaccine formulation with antigens derived from biofilms is a promising approach to prevent infectious diseases and can enhance the protective efficacy of existing vaccines. Therefore, research focusing on the identification of biofilm-derived antigens merits further investigations.

Stool Antigen Testing, a Reliable Noninvasive Method of Assessment of Helicobacter pylori Infection Among Patients with Gastro-duodenal Disorders in Cameroon.

BACKGROUND: Several techniques such as invasive and noninvasive are used for the diagnosis of H. pylori infection. AIM: The aim of this study was to compare the results of rapid urease test, stool antigen test and serology in diagnosing H. pylori infection in Cameroon. METHODS: Hundred patients (66 women and 34 men) were enrolled. Each patient gave a written consent. The study was approved by the local Ethical Committee of Medical Sciences and the institutional review board. From each patient, blood, stool and gastric biopsies samples were collected for H. pylori detection using three methods: stool antigen test, serology and rapid urease test (RUT), taken as gold standard. Statistical analysis was performed using Graph pad Prism 7. RESULTS: Helicobacter pylori infection was detected in 43%, 45% and 73% of patients based on the RUT, stool antigen test and serology, respectively. The difference was statistically significant between serology and RUT (P = 0.0026), but not between stool antigens test and RUT (P = 0.288). Taken RUT as gold standard, the sensitivity, specificity, positive and negative predictive values of stool antigens test and serology were 65.11, 70.17, 62.22 and 72.72%; 88.37, 40.35, 55.77 and 82.14%, respectively. The accuracy of stool antigen test and serology was 68 and 61%, respectively. CONCLUSIONS: Our finding showed that stool antigen test can be used as a noninvasive method of assessment of H. pylori infection in our setting. Serological test can be used in screening; however, further diagnostic tests need to be carried out to confirm seropositive cases.

Assessment of real-time PCR for Helicobacter pylori DNA detection in stool with co-infection of intestinal parasites: a comparative study of DNA extraction methods.

BACKGROUND: Many studies reported high prevalence of H. pylori infection among patients co-infected with intestinal parasites. Molecular approach for the DNA detection of those microbes in stool have been proposed. However there are a few reports that evaluated the effect of bead-beating in relation to the H. pylori outcome. Therefore, we developed and evaluated two TaqMan-based real-time PCR (rt-PCR) qualitative assays for the detection of ureC (glmM) and cagA of Helicobacter pylori on DNA extracted by three procedures. RESULTS: The two PCRs were analysed on 100 stool samples from patients who were screened for intestinal parasites. Three DNA extraction procedures were used: 1) automation with bead beating, 2) automation without bead beating and 3) hand column. The specificity of the new assays was confirmed by sequencing the PCR products and by the lack of cross-reactivity with other bacteria or pathogens DNA. Rt-PCR assays showed a detection limit of 10^4 bacteria/200 mg stool. The ureC_PCR with bead beating process was compared to conventional stool antigen test (SAT), with 94.12 and 93.75% of respectively sensitivity and specificity. However, the discordant samples were confirmed by DNA sequencing suggesting a potential higher sensitivity and specificity of PCR. CONCLUSIONS: Our findings showed that the automation with bead-beating -suggested procedure for intestinal parasitic infections- can reach highly sensitive results in H. pylori detection on stool compared also with SAT. Thus, this work can provide new insights into the practice of a clinical microbiology laboratory in order to optimize detection of gastro-intestinal infections. Further studies are needed to better define the clinical value of this technique.

A new recombinant F1 antigen as a cost and time-effective tool for plague diagnosis.

The Yersinia pestis capsular antigen F1 is widely used in plague laboratory diagnosis. Here, we describe the production of an F1 recombinant protein within reduced time and biosafety requirements. Its evaluation in hemagglutination tests indicated that the recombinant F1 can replace the conventional F1 protein for plague diagnosis.

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine, expressing the outer membrane protein OMP19 of Brucella species.

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.