Correlation between antibiotic susceptibilities and genotypes in Neisseria gonorrhoeae from different geographical origins: determinants monitoring by real- time PCR as a complementary tool for surveillance

ABSTRACT Objective To determine in Neisseria gonorrhoeae (NG) isolates from different geographical areas whether monitoring of major determinants involved in chromosomal antimicrobial resistance correlated with phenotypes and could constitute complementary tools for surveillance. Methods Real-time multiplex PCR assays targeting penA, mtrR, penB, ponA, gyrA and parC determinants were applied to 169 NG extracts. Minimum inhibitory concentrations for penicillin and ciprofloxacin were determined by E tests, and b-lactamase production was analysed using nitrocefin discs

Differential Cytokine Gene Expression According to Outcome in a Hamster Model of Leptospirosis

Leptospirosis is a widespread bacterial infection that is transmitted by soil or water contaminated by the urine of infected animals, or directly from these animals. It has highly diverse clinical presentations, making its differential diagnosis difficult. Though most cases are minor and self-resolving, there are also severe forms that include a sepsis pattern and multiple organ failure, and have possible fatal outcomes. Predictors of disease evolution and outcome are scarce, yet they would be very valuable to clinicians as well as to better decipher disease pathogenesis. In this study, we used a hamster model of leptospirosis to evaluate if immune genes were differentially expressed between individuals and if their expression levels could help forecast the outcome of the disease. We found that hamsters that later died from leptospirosis had significantly higher expression levels of both pro- and anti-inflammatory mediators compared to survivors. These results suggest that expression levels of these immune effectors might be helpful predictors of outcome in leptospirosis and that septic shock contributes to fatal leptospirosis

Escape of TLR5 Recognition by Leptospira spp.: A Rationale for Atypical Endoflagella

Leptospira (L.) interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagellae that allow their motility. L. interrogans are stealth pathogens that escape the innate immune recognition of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, which senses peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by qPCR. We did not identify any protective or inflammatory role of murine TLR5 for controlling pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5 signaling. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, but did not, or barely induced stimulation via murine TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, after using antimicrobial peptides to destabilize live leptospires, we detected TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in the flagellin proteins, we further showed that neither FlaA nor Fcp participated in the recognition by TLR5, suggesting a role for the FlaB. FlaB have structural homology to Salmonella FliC, and possess conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response

Production and Characterization of Chimeric Monoclonal Antibodies against Burkholderia pseudomallei and B. mallei Using the DHFR Expression System

Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection

Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis

Leptospirosis is a bacterial disease that is mainly spread by rodents and other small mammals. Transmission frequently occurs in (sub-) tropical countries, where environmental circumstances are most favourable. Severe leptospirosis can cause bleeding and vital organ dysfunction. An exaggerated immune response is thought to play an important role in the pathophysiology of leptospirosis. Soluble ST2 (sST2) is thought to inhibit negative regulatory pathways of this response. Soluble ST2 is produced by cells that surround, for example, blood vessels, and several of these blood cells play an important part in the host immune response. In an observational study, we measured the extent of sST2 release in patients suffering from severe leptospirosis. We found that patients that died from leptospirosis displayed higher levels of sST2. Moreover, from this study we have seen that sST2 levels were associated with bleeding, whereas other markers of infection were not. In an experiment, we showed that (white) blood cells did not seem to be the source of sST2 production. Damage to blood vessels is likely to cause bleeding in leptospirosis patients, exposing sST2 producing cells like fibroblasts to the blood stream. Hence, we believe that sST2 may be used as a marker for tissue damage in patients suffering from severe leptospirosis

Syrian Hamster as an Animal Model for the Study on Infectious Diseases

Infectious diseases still remain one of the biggest challenges for human health. In order to gain a better understanding of the pathogenesis of infectious diseases and develop effective diagnostic tools, therapeutic agents, and preventive vaccines, a suitable animal model which can represent the characteristics of infectious is required. The Syrian hamster immune responses to infectious pathogens are similar to humans and as such, this model is advantageous for studying pathogenesis of infection including post-bacterial, viral and parasitic pathogens, along with assessing the efficacy and interactions of medications and vaccines for those pathogens. This review summarizes the current status of Syrian hamster models and their use for understanding the underlying mechanisms of pathogen infection, in addition to their use as a drug discovery platform and provides a strong rationale for the selection of Syrian hamster as animal models in biomedical research. The challenges of using Syrian hamster as an alternative animal model for the research of infectious diseases are also addressed. Keywords: infectious diseases, Syrian hamster, drug discovery, infection mechanism, biomedical researc

Protease of cryptococcus neoformans : role during infection and potential as prognostic evaluation biomarker

La cryptococcose est une infection opportuniste grave, associée à un taux de mortalité élevé en dépit d’un traitement antifongique adéquat. Dans un modèle murin de cryptococcose disséminée, les souris survivantes à l'inoculation d'une dose habituellement mortelle de Cryptococcus neoformans (Cn) ont majoritairement développé une réponse humorale contre une aspartyl protéase (PEP1). Nous avons évalué si une vaccination avec rPep1 et/ou une sérothérapie avec des anticorps anti-PEP1 pouvaient modifier le cours de l'infection.Par comparaison à une mortalité de 100 % chez les témoins, la vaccination de souris avec la protéine recombinante rPep1 avant l'inoculation des Cn permettait de prolonger leur survie et de diminuer leur charge fongique. Le vaccin thérapeutique basé sur une seule injection de rPep1 chez des souris préalablement infectées a prolongé leur survie, avec un contrôle partiel à total de leur infection dans les tissus. Le traitement de souris avec 1 injection d’anti-PEP1 7 jours après inoculation des Cn a permis de prolonger leur survie (effet dépendant de la souche de Cn, de l’Acm testé, du moment et de la dose administrée) par rapport aux souris traitées avec un Acm non pertinent. Les différents anti-PEP1 testés reconnaissaient plusieurs épitopes, n'ont pas démontré de capacité opsonisantes mais ont pu influencer la croissance de C. neoformans. L'expression in vivo du gène PEP1 était différente selon la souche inoculée, quel que soit le stade de la maladie et nous avons prouvé que PEP1 était sécrété dans le compartiment extracellulaire en association avec le développement de l'infection, suggérant pour PEP1, un rôle de possible biomarqueurCryptococcosis is a severe opportunistic infection associated with a 20 % mortality rate despite adequate antifungal therapy. In a relevant murine model of disseminated cryptococcosis, mice that survive the inoculation of a usually lethal challenge with Cryptococcus neoformans (Cn) were more likely to develop a delayed and monospecific antibody response against an aspartyl protease (PEP1). We assessed whether vaccination with rPep1 and/or serotheray with anti-PEP1 antibodies could alter the course of the infection. Compared to 100 % mortality rate in control mice, active immunization with recombinant protein rPep1 prior to Cn inoculation was associated with prolonged survival and decreased fungal burden. Therapeutic vaccine based on a single injection of rPep1 in mice previously infected provided prolonged survival with partial to total control of the infection of tissues. Passive serotherapy with 1 injection of anti-PEP1 Mabs 7 days after Cn inoculation led to a prolonged survival (dependent on the Cn strain, the Mab tested, the timing and the Mab dose) compared to mice treated with an irrelevant Mab. Anti-PEP1 Mabs have recognized different epitopes, have not demonstrated opsonisation capacity but were able to impact the growth of C. neoformans. The in vivo expression of the gene PEP1 was different depending on the inoculated strain, irrespective of the stage of the disease and we proved that Pep1 was secreted into the extracellular compartment in association with the development of infection, suggesting a possible role as biomarker for PEP1. These results suggest that immunomodulation with PEP1 or anti-PEP1 may be of benefit during disseminated cryptococcosi

Recent findings related to immune responses against leptospirosis and novel strategies to prevent infection

International audienceWhat are the new approaches and emerging ideas to prevent leptospirosis, a neglected bacterial re-emerging zoonotic disease? How do Leptospira interrogans escape the host defenses? We aim here to review and discuss the most recent literature that provides some answers to these questions, in particular data related to a better understanding of adaptive and innate immunity towards leptospires, and design of vaccines. This is an opinion paper, not a comprehensive review. We will try to highlight the new strategies and technologies boosting the search for drugs and vaccines. We will also address the bottlenecks and difficulties impairing the search for efficient vaccines and the many gaps in our knowledge of immunity against leptospirosis. Finally, we aim to delineate how Leptospira spp. escape the innate immune responses of Toll-Like receptors (TLR) and Nod-Like receptors (NLR). The rational use of TLR and NLR agonists as adjuvants could be key to design future vaccines against pathogenic leptospires