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Burkholderia pseudomallei (Bpm) is the causative agent of the disease melioidosis. As a facultative intracellular pathogen, Bpm has a complex lifestyle that culminates in cell-to-cell fusion and multinucleated giant cells (MNGCs) formation. The virulence factor responsible for MNGC formation is the type 6 secretion system (T6SS), a contractile nanomachine. MNGC formation is a cell-to-cell spread strategy that allows the bacteria to avoid the extracellular immune system and our previous data highlighted cell death, apoptosis, and inflammation as pathways significantly impacted by T6SS activity. Thusly, we investigated how the T6SS influences these phenotypes within the macrophage and pulmonary models of infection. Here we report that the T6SS is responsible for exacerbating apoptotic cell death during infection in both macrophages and the lungs of infected mice. We also demonstrate that although the T6SS does not influence differential macrophage polarization, the M2 polarization observed is potentially beneficial for Bpm pathogenesis and replication. Finally, we show that the T6SS contributes to the severity of inflammatory nodule formation in the lungs, which might be potentially connected to the amount of apoptosis that is triggered by the bacteria. Author summary: Burkholderia pseudomallei (Bpm) is an intracellular pathogen and is the etiological agent of melioidosis. This neglected tropical disease results in an estimated 89,000 fatalities a year, however, this number is believed to be severely underreported. The complex intracellular lifestyle and host response to infection is poorly understood, basic cell interactions and responses are critical to defense against infection. Bpm utilizes an array of virulence factors to successfully replicate and disseminate cell-to-cell, one of these is the T6SS which is responsible for MNGC formation and cell-to-cell spread. In this work, we characterized how macrophages respond to infection in the presence or absence of the critical T6SS virulence factor. Macrophages are a primary replicative niche for Bpm but how intracellular replication disrupts macrophages response to infection is poorly understood. By understanding macrophage cell death patterns and polarization, we can dissect responses that are triggered by T6SS activity with the expectation of finding exploitable responses for host directed therapies. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic E. coli (EHEC) is a group of pathogenic bacteria that is associated with worldwide human foodborne diarrheal illnesses and the development of hemolytic uremic syndrome, a potentially deadly condition associated with Shiga toxins (Stxs). Currently, approved vaccines for human prophylaxis against infection do not exist, and one barrier preventing the successful creation of EHEC vaccines is the absence of dependable animal models, including mice, which are naturally resistant to EHEC infection and do not manifest the characteristic signs of the illness. Our lab previously developed gold nanoparticle (AuNP)-based EHEC vaccines, and assessed their efficacy using Citrobacter rodentium, which is the mouse pathogen counterpart of EHEC, along with an Stx2d-producing strain that leads to more consistent disease kinetics in mice, including lethality. The purpose of this study was to continue evaluating these vaccines to increase protection. Here, we demonstrated that subcutaneous immunization of mice with AuNPs linked to the EHEC antigens EscC and intimin (Eae), either alone or simultaneously, elicits functional robust systemic humoral responses. Additionally, vaccination with both antigens together showed some efficacy against Stx2d-producing C. rodentium while AuNP-EscC successfully limited infection with non-Stx2d-producing C. rodentium. Overall, the collected results indicate that our AuNP vaccines have promising potential for preventing disease with EHEC, and that evaluation of novel vaccines using an appropriate animal model, like C. rodentium described here, could be the key to finally developing an effective EHEC vaccine that can progress into human clinical trials. [ABSTRACT FROM AUTHOR]

We investigated bile salts' ability to induce phenotypic changes in biofilm production and protein expression of pathogenic Escherichia coli strains. For this purpose, 82 pathogenic E. coli strains isolated from humans (n  = 70), and animals (n  = 12), were examined for their ability to form biofilms in the presence or absence of bile salts. We also identified bacterial proteins expressed in response to bile salts using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-electrophoresis) and liquid chromatography-mass spectrometry (LC-MS/MS). Lastly, we evaluated the ability of these strains to adhere to Caco-2 epithelial cells in the presence of bile salts. Regarding biofilm formation, two strains isolated from an outbreak in Republic of Georgia in 2009 were the only ones that showed a high and moderate capacity to form biofilm in the presence of bile salts. Further, we observed that those isolates, when in the presence of bile salts, expressed different proteins identified as outer membrane proteins (i.e. OmpC), and resistance to adverse growth conditions (i.e. F0F1, HN-S, and L7/L12). We also found that these isolates exhibited high adhesion to epithelial cells in the presence of bile salts. Together, these results contribute to the phenotypic characterization of E. coli O104: H4 strains. [ABSTRACT FROM AUTHOR]

Burkholderia are a group of Gram-negative bacteria that can cause a variety of diseases in at-risk populations. B. pseudomallei and B. mallei, the etiological agents of melioidosis and glanders, respectively, are the two clinically relevant members of the B. pseudomallei complex (Bpc). The development of vaccines against Bpc species has been accelerated in recent years, resulting in numerous promising subunits and glycoconjugate vaccines incorporating a variety of antigens. However, a second group of pathogenic Burkholderia species exists known as the Burkholderia cepacia complex (Bcc), a group of opportunistic bacteria which tend to affect individuals with weakened immunity or cystic fibrosis. To date, there have been few attempts to develop vaccines to Bcc species. Therefore, the primary goal of this review is to provide a broad overview of the various subunit antigens that have been tested in Bpc species, their protective efficacy, study limitations, and known or suspected mechanisms of protection. Then, we assess the reviewed Bpc antigens for their amino acid sequence conservation to homologous proteins found in Bcc species. We propose that protective Bpc antigens with a high degree of Bpc-to-Bcc sequence conservation could serve as components of a pan-Burkholderia vaccine capable of protecting against both disease-causing groups. [ABSTRACT FROM AUTHOR]

Recently, the pathogen that causes melioidosis, Burkholderia pseudomallei, was found in the Gulf Coast region of Mississippi, United States of America, associated with human cases and as bacteria in the soil of affected areas. Therefore, the Centers for Disease Control and Prevention has declared the pathogen as endemic in the continental United States for the first time. This viewpoint discusses some issues that the research, public health communities, and government agencies need to address. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease 2019 (COVID-19). The high rate of mutation of this virus is associated with a quick emergence of new viral variants that have been rapidly spreading worldwide. Several mutations have been documented in the receptor-binding domain (RBD) of the viral spike protein that increases the interaction between SARS-CoV-2 and its cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Mutations in the spike can increase the viral spread rate, disease severity, and the ability of the virus to evade either the immune protective responses, monoclonal antibody treatments, or the efficacy of current licensed vaccines. This review aimed to highlight the functional virus classification used by the World Health Organization (WHO), Phylogenetic Assignment of Named Global Outbreak (PANGO), Global Initiative on Sharing All Influenza Data (GISAID), and Nextstrain, an open-source project to harness the scientific and public health potential of pathogen genome data, the chronological emergence of viral variants of concern (VOCs) and variants of interest (VOIs), the major findings related to the rate of spread, and the mutations in the spike protein that are involved in the evasion of the host immune responses elicited by prior SARS-CoV-2 infections and by the protection induced by vaccination. [ABSTRACT FROM AUTHOR]

Melioidosis, caused by Burkholderia pseudomallei (Bpm), lacks a vaccine. We identify the immune correlates of protection induced by B. mallei ΔtonB Δhcp1 (CLH001) and Bpm ΔtonB Δhcp1 (PBK001) vaccines against inhalational melioidosis. Mucosal immunization with either vaccine generates Bpm-specific IgM and IgG (IgG2b/c > IgG1 > IgG3) antibodies in sera and lungs, and lung IgA antibodies. Sera confers complement-independent bactericidal activity and macrophages opsonophagocytic uptake but is insufficient in passive transfer experiments to provide significant protection. Both vaccines elicit memory Th1 and Th17 CD4+ T-cell responses in lung and spleen after Bpm antigen-specific recall. The PBK001 vaccine is superior in generating respiratory IgA post-boost, anamnestic IgG at challenge, T-cell recall to specific antigen, and development of diverse polyfunctional memory T-cell pools. Analysis of lung histology suggests that potent polyfunctional T-cell memory and/or IL-17 signatures generated with PBK001 vaccination may be associated with moderate lung inflammation post vaccination. [ABSTRACT FROM AUTHOR]

Background: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In Mexico, the disease is rarely diagnosed in humans and there is no evidence of simultaneous environmental isolation of the pathogen. Here, we describe clinical profiles of fatal cases of melioidosis in two children, in a region without history of that disease.Case Presentation: About 48 h before onset of symptoms, patients swam in a natural body of water, and thereafter they rapidly developed fatal septicemic illness. Upon necropsy, samples from liver, spleen, lung, cerebrospinal fluid, and bronchial aspirate tissues contained Burkholderia pseudomallei. Environmental samples collected from the locations where the children swam also contained B. pseudomallei. All the clinical and environmental strains showed the same BOX-PCR pattern, suggesting that infection originated from the area where the patients were swimming.Conclusions: The identification of B. pseudomallei confirmed that melioidosis disease exists in Sonora, Mexico. The presence of B. pseudomallei in the environment may suggest endemicity of the pathogen in the region. This study highlights the importance of strengthening laboratory capacity to prevent and control future melioidosis cases. [ABSTRACT FROM AUTHOR]

We report an analysis of the genomic diversity of isolates of Burkholderia pseudomallei, the cause of melioidosis, recovered in Colombia from routine surveillance during 2016-2017. B. pseudomallei appears genetically diverse, suggesting it is well established and has spread across the region. [ABSTRACT FROM AUTHOR]

is an environmental intracellular Gram-negative bacterium that causes melioidosis, a severe infectious disease affecting humans and animals. An increase in melioidosis cases worldwide and the high mortality rate of the disease makes it a public health concern. Melioidosis is known as the 'great mimicker' because it presents with a wide range of disease manifestations. B. pseudomallei is naturally resistant to antibiotics and delay in diagnosis leads to ineffective treatment. Furthermore, there is no approved vaccine to prevent melioidosis infection in humans. Therefore, it is a priority to license a vaccine that can be used for both high-risk endemic areas and for biodefense purposes. In this review, we have focussed on recent progress in the USA for the development and advancement of lead B. pseudomallei vaccine candidate(s) ready for testing in pre-clinical trials. Those candidates include live-attenuated vaccines, glycoconjugate vaccines, outer-membrane vesicles, and gold nanoparticle vaccines. Side-by-side comparison of the leading B. pseudomallei vaccine candidates will provide important information to further advance studies into pre-clinical trials. The likelihood of any of these current vaccines becoming the selected candidate that will reduce the occurrence of melioidosis worldwide is closer than ever. [ABSTRACT FROM AUTHOR]

Background: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strain in the susceptible BALB/c mouse model. Methodology/Principal finding: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. Conclusions/Significance: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies. [ABSTRACT FROM AUTHOR]

Background: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strain s in the susceptible BALB/c mouse model. Methodology/Principal finding: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. Conclusions/Significance: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies. Author summary: Glanders (caused by Burkholderia. mallei) and melioidosis (caused by B. pseudomallei) are severe infectious diseases of concern worldwide because of the rising number of cases and mortality rate. The low infectious doses of these two pathogens along with their amenability for aerosolization are factors that could be exploited as potential biothreat agents. Once the diseases have developed in humans and animals, intrinsic resistance to broad classes of antibiotics becomes a challenge for treatment and increases the risk for relapse. The progress in vaccine development demonstrates that live attenuated vaccine strains are the most effective in protection and providing long-lasting immune responses against both diseases. Our data indicate that the B. mallei double mutant (ΔtonB Δhcp1) strain CLH001, is a feasible vaccine candidate to prevent glanders and melioidosis, especially for biodefense and public health purposes. [ABSTRACT FROM AUTHOR]

The article discusses care of melioidosis patients caused by burkholderia pseudomallei, antibiotics resistance and synergistic effect of aminoglycosides. Topics include use of polymerase chain reaction test, biochemical tests with involving resistance to gentamicin and colistin and hemagglutination test.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease that requires long-term treatment regimens with no assurance of bacterial clearance. Clinical isolates are intrinsically resistant to most antibiotics and in recent years, isolates have been collected that display resistance to frontline drugs. With the expanding global burden of B. pseudomallei, there is a need to identify new compounds or improve current treatments to reduce risk of relapse. Using the Pathogen Box generated by Medicines for Malaria Venture, we screened a library of 400 compounds for bacteriostatic or bactericidal activity against B. pseudomallei K96243 and identified seven compounds that exhibited inhibitory effects. New compounds found to have function against B. pseudomallei were auranofin, rifampicin, miltefosine, MMV688179, and MMV688271. An additional two compounds currently used to treat melioidosis, doxycycline and levofloxacin, were also identified in the screen. We determined that the minimal inhibitory concentrations (MIC) for levofloxacin, doxycycline, and MMV688271 were below 12 μg/ml for 5 strains of B. pseudomallei. To assess persister frequency, bacteria were exposed to 100x MIC of each compound. Auranofin, MMV688179, and MMV688271 reduced the bacterial population to an average of 4.53 × 10−6% compared to ceftazidime, which corresponds to 25.1% survival. Overall, our data demonstrates that auranofin, MMV688197, and MMV688271 have the potential to become repurposed drugs for treating melioidosis infections and the first evidence that alternative therapeutics can reduce B. pseudomallei persistence. [ABSTRACT FROM AUTHOR]

Burkholderia cenocepacia is an opportunistic pathogen prevalent in cystic fibrosis patients, which is particularly difficult to treat, causing chronic and eventually fatal infections. The lack of effective treatment options makes evident the need to develop alternative therapeutic or prophylactic approaches. Vaccines, and live attenuated vaccines, are an unexplored avenue to treat B. cenocepacia infections. Here we constructed and characterized a B. cenocepacia tonB mutant strain, which was unable to actively transport iron, to test whether this single gene deletion mutant (strain renamed GAP001) protected against an acute respiratory B. cenocepacia lethal infection. Here we show that the mutant strain GAP001 is attenuated, and effective at protecting against B. cenocepacia challenge. Intranasal administration of GAP001 to BALB/c mice resulted in almost complete survival with high degree of bacterial clearance. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains. [ABSTRACT FROM AUTHOR]

The emergence and evolution of pathogenic Escherichia coli strains associated with animal and human infections continues being a topic of active investigation in recent years. Latin America has responded to this thread establishing the Latin American Coalition for Escherichia coli Research (LACER), a multidisciplinary group of scientists applying principles of the One Health initiative. LACER is integrating animal, human and environmental health for the mutual benefit of all, while reducing the impact of human disease. Here, I depict the usefulness of this approach by providing three examples from several priority areas in E. coli research and comparing them to the activities performed by LACER, which are enhancing collaborative efforts in biomedical research and providing a translational platform to connect with epidemiological studies and public health policy makers. The manuscript concludes with a brief historical description regarding the establishment of LACER, emphasizing the importance of collaboration between health professionals of different international organizations and not only across one specific country, to effectively combat current and future E. coli infections. The benefits of utilizing 'One Health' concepts in the establishment of LACER should encourage other professionals studying zoonotic or environmentally acquired human infections to implement effective international collaborations to combat common problems. [ABSTRACT FROM AUTHOR]

Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous strains in terms of serotypes, adherence patterns and the presence of novel virulence factors. This heterogeneity is intriguing, promoting studies trying to characterize these novel proteins and to better comprehend this pathotype group. In a previous study analyzing low-molecular mass proteomes of four representative aEPEC strains of three different adhesion phenotypes, we classified proteins according to their annotated function, with most of them being involved in metabolism and transport; while some of them were classified as hypothetical proteins. The majority of the hypothetical proteins were homologue products of genes identified in the genome of enterohemorrhagic E. coli. One of the hypothetical proteins was annotated as Z2335, with orthologue in EPEC, and by bioinformatics analysis, this protein was revealed to be the universal stress protein F (UspF). Thus, herein we successfully obtained a recombinant UspF protein from aEPEC, which is a α/β, ATP-binding protein involved in stress response, with comparable protein production among the four studied strains, but showing noteworthy differences when cultivated in different stress conditions, also present in other enterobacterial species, such as Shigella sonnei and Citrobacter freundii. Furthermore, our results confirm that the Usp protein superfamily encompasses a conserved group of proteins involved in stress resistance in aEPEC and other Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

Escherichia colibiotype O104:H4 recently caused the deadliestE. colioutbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impactin vivocolonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as ‘Generally Recognized as Safe’ (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks. [ABSTRACT FROM PUBLISHER]

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a leading cause of foodborne illnesses worldwide and is a common serotype linked to hemorrhagic colitis and an important cause of hemolytic uremic syndrome (HUS). Treatment of EHEC O157:H7 infections is complicated, as antibiotics can exacerbate Shiga toxin (Stx) production and lead to more severe symptoms including HUS. To date, no vaccines have been approved for human use, exposing a void in both treatment and prevention of EHEC O157:H7 infections. Previously, our lab has shown success in identifying novel vaccine candidates via bio- and immunoinformatics approaches, which are capable of reducing bacterial colonization in an in vivo model of intestinal colonization. In this study, we further characterized 17 of the identified vaccine candidates at the bioinformatics level and evaluated the protective capacity of the top three candidates when administered as DNA vaccines in our murine model of EHEC O157:H7 colonization. Based on further immunoinformatic predictions, these vaccine candidates were expected to induce neutralizing antibodies in a Th2-skewed immunological response. Immunization of BALB/c mice with two of these candidates resulted in reduced bacterial colonization following EHEC O157:H7 challenge. Additionally, immune sera was shown to prevent bacterial adhesion in vitro to Caco-2 cells. Together, this study provides further validation of our immunoinformatic analyses and identifies promising vaccine candidates against EHEC O157:H7. [ABSTRACT FROM AUTHOR]

The Gram-negative proteobacteria genus Burkholderia encompasses multiple bacterial species that are pathogenic to humans and other vertebrates. Two pathogenic species of interest within this genus are Burkholderia pseudomallei (Bpm) and the B. cepacia complex (Bcc); the former is the causative agent of melioidosis in humans and other mammals, and the latter is associated with pneumonia in immunocompromised patients. One understudied and shared characteristic of these two pathogenic groups is their ability to persist and establish chronic infection within the host. In this review, we will explore the depth of knowledge about chronic infections caused by persistent Bpm and Bcc. We examine the host risk factors and immune responses associated with more severe chronic infections. We also discuss host adaptation and phenotypes associated with persistent Burkholderia species. Lastly, we survey how other intracellular bacteria associated with chronic infections are combatted and explore possible future applications to target Burkholderia. Our goal is to highlight understudied areas that should be addressed for a more thorough understanding of chronic Burkholderia infections and how to combat them. [ABSTRACT FROM AUTHOR]

A renewed interest in Shiga toxin-producing Escherichia coli (STEC) strains was sparked due to the appearance of an outbreak in 2011, causing 3,816 diarrheal cases and some deaths in Europe. The causative strain was classified as enteroaggregative E. coli of serotype O104:H4 that had acquired Shiga toxin genes. The ability of STEC O104:H4 to cause disease relies greatly on the bacteria’s capacity to colonize, persist, and produce Shiga toxin. However, not much is known about the colonization factors of this strain. Because long polar fimbriae (lpf) lpf1 and lpf2 operons encode important colonization factors in other STEC isolates and E. coli O104:H4 possesses both loci, we hypothesized that Lpf is required for adhesion and colonization. In this study, isogenic lpfA1 and lpfA2 major fimbrial subunit mutants were constructed. To determine their role in O104:H4’s virulence, we assessed their ability to adhere to non-polarized and polarized intestinal epithelial cells. The ΔlpfA1 showed decreased adherence in both cell systems, while the ΔlpfA2 only showed a decrease in adherence to polarized Caco-2 cells. We also tested the O104:H4 mutants’ ability to form biofilm and found that the ΔlpfA1 was unable to form a stable biofilm. In an in vivo murine model of intestinal colonization, the ΔlpfA1 had a reduced ability to colonize the cecum and large intestine, consistent with the in vitro data. Further, we tested the lpfA1 mutants’ ability to compete against the wild type. We found that in the in vitro and in vivo models, the presence of the wild type O104:H4 facilitates increased adherence of the ΔlpfA1 to levels exceeding that of the wild type. Overall, our data demonstrated that Lpf1 is one of the factors responsible for O104:H4 intestinal adhesion and colonization. [ABSTRACT FROM AUTHOR]

Background: In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Methodology/Principal Findings: Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Conclusions/Significance: Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis. Author Summary: Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively. In addition to the recent rise in cases of glanders and the endemicity of melioidosis worldwide, these pathogens have gained attention as potential bioweapons. Further, these pathogens have huge potential for aerosol delivery and often produce fatal infection amongst untreated individuals. Both pathogens are difficult to treat, and even with antibiotic intervention, patients relapse or get re-infected. A big challenge for vaccine development against these pathogens includes identification of broadly protective antigens and a better understanding of the correlates of protection from both acute and chronic infections. Our study is the first to demonstrate significant protection against a lethal challenge with both Burkholderia species. Because TMM001 persists in immunized mice, we propose that this attenuated organism is a promising backbone-based strain from which a legitimate vaccine candidate can be generated. [ABSTRACT FROM AUTHOR]

Background: In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Methodology/Principal Findings: Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Conclusions/Significance: Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis. [ABSTRACT FROM AUTHOR]

Several candidates for a vaccine against Burkholderia pseudomallei, the causal bacterium of melioidosis, have been developed, and a rational approach is now needed to select and advance candidates for testing in relevant nonhuman primate models and in human clinical trials. Development of such a vaccine was the topic of a meeting in the United Kingdom in March 2014 attended by international candidate vaccine developers, researchers, and government health officials. The focus of the meeting was advancement of vaccines for prevention of natural infection, rather than for protection from the organism's known potential for use as a biological weapon. A direct comparison of candidate vaccines in well-characterized mouse models was proposed. Knowledge gaps requiring further research were identified. Recommendations were made to accelerate the development of an effective vaccine against melioidosis. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) strains are causative agents of diarrhea and hemorrhagic colitis, both diseases associated with intestinal inflammation and cell damage. Several studies have correlated EHEC virulence factors to high levels of intestinal pro-inflammatory cytokines and we have previously described that the Long polar fimbriae (Lpf) is involved in the secretion of interleukin-8 (IL-8) and up-regulation of genes belonging to the NF-κB pathway using non-polarized epithelial intestinal T84 cells. In the current study, we evaluated the two EHEC O157 Lpf fimbriae (Lpf1 and Lpf2) for their ability to induce intestinal secretion of IL-8 and the activation of IL8, CCL20, and ICAM1 genes on polarized T84 cells.We also determined the participation of Lpf1 and Lpf2 in transepithelial migration of polymorphonuclear neutrophils (PMNs). Polarized T84 cells infected with EHEC revealed that both, Lpf1 and Lpf2, were required for the secretion of IL-8 and the induction of IL8, CCL20, and ICAM1 genes. Both fimbriae also played a role in the migration of PMNs trough the intestinal cells monolayer. Overall, the present work further demonstrated that the fimbriae Lpf1 and Lpf2 are important bacterial virulence factors that might be involved in the inflammatory responses associated with EHEC infections. [ABSTRACT FROM AUTHOR]

The article discusses the importance of the international collaboration in advancing the research endeavors. Topics mention including the globalization in the food industry and its impact on the agricultural trade, requiring multidisciplinary approach in order to such as identify, characterize and diagnose a certain pathogen and formation of Latin America Coalition for E. coli Research(LACER).

The molecular mechanisms controlling expression of the long polar fimbriae 2 (Lpf2) of enterohemorrhagic Escherichia coli ( EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT- PCR demonstrated that the highest lpfA2 expression occurs between an OD600 of 1.0 and 1.2 in DMEM at pH 6.5 and 37 °C. The level of lpfA2 transcription at OD600 1.2 and pH 6.5 was four times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2 times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25 °C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7 times more abundant than baseline conditions. Further, transcription in the EDL933∆ fur was 0.6 and 0.8 times higher as compared with the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during the exponential phase of growth, and is controlled by Fur. [ABSTRACT FROM AUTHOR]

Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens. [ABSTRACT FROM AUTHOR]

Long polar fimbriae ( Lpf) are intestinal adhesins and important virulence factors of pathogenic Escherichia coli strains. We cloned and sequenced the lpf2-1 operon ( lpf2ABCD ) and its flanking regions of an intimin- and Shiga toxin-negative atypical E. coli O157: H43 strain of bovine origin and also sequenced the lpf2-1 operon of six additional atypical O157 bovine E. coli strains of various serotypes. Nucleotide sequence comparison of these lpf operons showed sequence conservation as they contained only four polymorphic nucleotide positions. Investigation of these O157 strains as well as 13 E. coli Reference Collection ( ECOR) strains carrying the lpf2-1 allele revealed high degree of sequence conservation in the lpf2 flanking regions. The lpf2-1 allele is also present in a bovine Shiga toxin-producing E. coli STEC O136: H12 strain, and in vitro adherence assays revealed that the absence of lpf2-1 in this strain did not affect its host cell-binding properties. Our data indicate that lpf2 loci are highly conserved in E. coli isolates; however, its role in adherence might be masked by other uncharacterized adhesins. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone. [ABSTRACT FROM AUTHOR]

Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs) is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3), while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines postinfection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of inflammatory cell populations, despite a robust production of pro-inflammatory cytokines, was associated with poorly controlled bacterial growth, severe lung pathology, and death of control animals. [ABSTRACT FROM AUTHOR]

The intestinal epithelium provides a barrier between a variety of luminal antigens and provides the components of intestinal innate and adaptive immunity. It is crucial that at this interface, the epithelial cell layer and the components of the intestinal immunity interact with dietary and bacterial antigens in a regulated way to maintain homeostasis. Failure to tightly control immune reactions can be detrimental and result in inflammation. In the current review, we described the regulatory mechanisms controlling host-immune homeostasis and the role of regulatory CD4(+) T cells, with a special emphasis in the regulatory T-cell subsets (Tregs). Furthermore, the participation of innate cell cross-talk in the polarization of intestinal immune responses is also evaluated. Finally, the recent characterization of host responses to normal commensal flora, the role of bacteria and bacterial factors in the maintenance of immunomodulation, and the disruption of this balance by bacterial enteric pathogens is also summarized. [ABSTRACT FROM AUTHOR]

Background: A major outbreak of bloody diarrhea associated with Shiga toxin-producing Escherichia coli O104:H4 occurred early in 2011, to which an unusual number of hemolytic uremic syndrome cases were linked. Due to limited information regarding pathogenesis and/or virulence properties of this particular serotype, we investigated the contribution of the aerobactin iron transport system during in vitro and in vivo conditions. Results: A bioluminescent reporter construct was used to perform real-time monitoring of E. coli O104:H4 in a mouse model of infection. We verified that our reporter strain maintained characteristics and growth kinetics that were similar to those of the wild-type E. coli strain. We found that the intestinal cecum of ICR (CD-1) mice was colonized by O104:H4, with bacteria persisting for up to 7 days after intragastric inoculation. MALDI-TOF analysis of heat-extracted proteins was performed to identify putative surface-exposed virulence determinants. A protein with a high similarity to the aerobactin iron receptor was identified and further demonstrated to be up-regulated in E. coli O104:H4 when grown on MacConkey agar or during iron-depleted conditions. Because the aerobactin iron acquisition system is a key virulence factor in Enterobacteriaceae, an isogenic aerobactin receptor (iutA) mutant was created and its intestinal fitness assessed in the murine model. We demonstrated that the aerobactin mutant was out-competed by the wild-type E. coli O104:H4 during in vivo competition experiments, and the mutant was unable to persist in the cecum. Conclusion: Our findings demonstrate that bioluminescent imaging is a useful tool to monitor E. coli O104:H4 colonization properties, and the murine model can become a rapid way to evaluate bacterial factors associated with fitness and/or colonization during E. coli O104:H4 infections. [ABSTRACT FROM AUTHOR]

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches. [ABSTRACT FROM AUTHOR]

The long polar fimbriae (Lpf) is one of few adhesive factors of Shiga toxin-producing Escherichia coli (STEC) and it is associated with colonization of the intestine. Studies have demonstrated the presence of lpf genes in several pathogenic E. coli strains, and classification of variants based on polymorphisms in the lpfA1 and lpfA2 genes has been adopted. Using a collection of Argentinean locus of enterocyte effacement (LEE)-negative STEC strains, we determined that the different lpfA types were present in a wide variety of serotypes with no apparent association between the types of lpfA1 or lpfA2 genes and the severity of human disease. The lpfA2-1 was the most prevalent variant identified, which was present in 95.8% of the isolates, and lpfA1-3 and lpfA2-2, proposed as specific biomarkers of E. coli O157:H7, were not found in any of the serotypes studied. The prevalence of lpf genes in a large number of strains is useful to understand the genetic diversity of LEE-negative STEC and to define the association of some of these isolates carrying specific lpf-variants with disease. [ABSTRACT FROM AUTHOR]

Background: Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. Results: We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. Conclusions: Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease. [ABSTRACT FROM AUTHOR]

Background: Burkholderia mallei is a zoonotic Gram negative bacterium which primarily infects solipeds but can cause lethal disease in humans if left untreated. The effect of two antibiotics with different modes of action on Burkholderia mallei strain ATCC23344 was investigated by using in vitro and in vivo studies. Results: Determination of minimal inhibitory concentrations (MICs) in vitro was done by the agar diffusion method and the dilution method. The MICs of levofloxacin and ceftazidime were in the similar range, 2.5 and 5.0 µg/ml, respectively. Intracellular susceptibility of the bacterium to these two antibiotics in J774A.1 mouse macrophages in vitro was also investigated. Macrophages treated with antibiotics demonstrated uptake of the drugs and reduced bacterial loads in vitro. The efficacy of ceftazidime and levofloxacin were studied in BALB/c mice as post-exposure treatment following intranasal B. mallei infection. Intranasal infection with 5 x 105 CFUs of B. mallei resulted in 90% death in non-treated control mice. Antibiotic treatments 10 days post-infection proved to be effective in vivo with all antibiotic treated mice surviving to day 34 post-infection. The antibiotics did not result in complete clearance of the bacterial infection and presence of the bacteria was found in lungs and spleens of the survivors, although bacterial burden recovered from levofloxacin treated animals appeared reduced compared to ceftazidime. Conclusion: Both antibiotics demonstrated utility for the treatment of glanders, including the ability for intracellular penetration and clearance of organisms in vitro. [ABSTRACT FROM AUTHOR]