The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media

AbstractCoxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

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In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04324-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2113-2121 ◽

Cited By ~ 9

Author(s):

U. Malik ◽

O. N. Silva ◽

I. C. M. Fensterseifer ◽

L. Y. Chan ◽

R. J. Clark ◽

...

Keyword(s):

Antimicrobial Agents ◽

Cyclic Peptide ◽

Sequence Similarity ◽

Infection Model ◽

Content Type ◽

Wide Range ◽

Inhibitory Activities ◽

The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

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Coxiella burnetii Interaction with Neutrophils and MacrophagesIn Vitroand in SCID Mice following Aerosol Infection

Infection and Immunity ◽

10.1128/iai.00973-13 ◽

2013 ◽

Vol 81 (12) ◽

pp. 4604-4614 ◽

Cited By ~ 18

Author(s):

Alexandra Elliott ◽

Ying Peng ◽

Guoquan Zhang

Keyword(s):

Immune Response ◽

Coxiella Burnetii ◽

Q Fever ◽

Natural Infection ◽

Scid Mice ◽

Innate Immune ◽

Content Type ◽

Aerosol Infection

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium that causes acute and chronic Q fever in humans. Human Q fever is mainly transmitted by aerosol infection. However, there is a fundamental gap in the knowledge regarding the mechanisms of pulmonary immunity againstC. burnetiiinfection. This study focused on understanding the interaction betweenC. burnetiiand innate immune cellsin vitroandin vivo. Both virulentC. burnetiiNine Mile phase I (NMI) and avirulent Nine Mile phase II (NMII) were able to infect neutrophils, while the infection rates were lower than 29%, suggesting thatC. burnetiican infect neutrophils, but infection is limited. Interestingly,C. burnetiiinside neutrophils can infect and replicate within macrophages, suggesting that neutrophils cannot killC. burnetiiandC. burnetiimay be using infection of neutrophils as an evasive strategy to infect macrophages. To elucidate the mechanisms of the innate immune response toC. burnetiinatural infection, SCID mice were exposed to aerosolizedC. burnetii. Surprisingly, neutrophil influx into the lungs was delayed until day 7 postinfection in both NMI- and NMII-infected mice. This result suggests that neutrophils may play a unique role in the early immune response against aerosolizedC. burnetii. Studying the interaction betweenC. burnetiiand the innate immune system can provide a model system for understanding how the bacteria evade early immune responses to cause infection.

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Pathogenetic Impact of Bacterial–Fungal Interactions

Microorganisms ◽

10.3390/microorganisms7100459 ◽

2019 ◽

Vol 7 (10) ◽

pp. 459 ◽

Cited By ~ 8

Author(s):

Filomena Nogueira ◽

Shirin Sharghi ◽

Karl Kuchler ◽

Thomas Lion

Keyword(s):

Immune System ◽

Clinical Manifestations ◽

Antimicrobial Treatment ◽

Human Pathogens ◽

Human Fungal Pathogen ◽

Bacteria And Fungi ◽

Fungal Interactions ◽

The Impact

Polymicrobial infections are of paramount importance because of the potential severity of clinical manifestations, often associated with increased resistance to antimicrobial treatment. The intricate interplay with the host and the immune system, and the impact on microbiome imbalance, are of importance in this context. The equilibrium of microbiota in the human host is critical for preventing potential dysbiosis and the ensuing development of disease. Bacteria and fungi can communicate via signaling molecules, and produce metabolites and toxins capable of modulating the immune response or altering the efficacy of treatment. Most of the bacterial–fungal interactions described to date focus on the human fungal pathogen Candida albicans and different bacteria. In this review, we discuss more than twenty different bacterial–fungal interactions involving several clinically important human pathogens. The interactions, which can be synergistic or antagonistic, both in vitro and in vivo, are addressed with a focus on the quorum-sensing molecules produced, the response of the immune system, and the impact on clinical outcome.

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Protein Binding: Do We Ever Learn?

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01433-10 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3067-3074 ◽

Cited By ~ 143

Author(s):

Markus A. Zeitlinger ◽

Hartmut Derendorf ◽

Johan W. Mouton ◽

Otto Cars ◽

William A. Craig ◽

...

Keyword(s):

Protein Binding ◽

Protein Concentration ◽

Antimicrobial Agents ◽

Quantitative Relationship ◽

New Antibiotics ◽

Protein Rich ◽

High Degree ◽

The Impact

ABSTRACTAlthough the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agentsin vitro. This might explain the somewhat contradictory results obtained from different studies. Simplein vitromodels which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Withinin vitrotest systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibioticsin vitrobecome even more urgent. In addition, the quantitative relationship between the effects of protein bindingin vitroandin vivoneeds to be established, since the physiological conditions differ. General recommendations for testing the impact of protein bindingin vitroare suggested.

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Avoidance of the NLRP3 Inflammasome by the Stealth Pathogen, Coxiella burnetii

Veterinary Pathology ◽

10.1177/0300985820981369 ◽

2020 ◽

pp. 030098582098136

Author(s):

Martha A. Delaney ◽

Andreas den Hartigh ◽

Samuel J. Carpentier ◽

Timothy P. Birkland ◽

Donald P. Knowles ◽

...

Keyword(s):

Coxiella Burnetii ◽

Q Fever ◽

Bacterial Pathogen ◽

In Vivo Model ◽

Caspase 1 ◽

Type Iv ◽

Clinical Resistance ◽

Nlrp3 Inflammasomes

Coxiella burnetii, a highly adapted obligate intracellular bacterial pathogen and the cause of the zoonosis Q fever, is a reemerging public health threat. C. burnetii employs a Type IV secretion system (T4SS) to establish and maintain its intracellular niche and modulate host immune responses including the inhibition of apoptosis. Interactions between C. burnetii and caspase-1-mediated inflammasomes are not fully elucidated. This study confirms that C. burnetii does not activate caspase-1 during infection of mouse macrophages in vitro. C. burnetii–infected cells did not develop NLRP3 and ASC foci indicating its ability to avoid cytosolic detection. C. burnetii is unable to inhibit the pyroptosis and IL-1β secretion that is induced by potent inflammasome stimuli but rather enhances these caspase-1-mediated effects. We found that C. burnetii upregulates pro-IL-1β and robustly primes NLRP3 inflammasomes via TLR2 and MyD88 signaling. As for wildtype C. burnetii, T4SS-deficient mutants primed and potentiated NLRP3 inflammasomes. An in vivo model of pulmonary infection in C57BL/6 mice was developed. Mice deficient in NLRP3 or caspase-1 were like wildtype mice in the development and resolution of splenomegaly due to red pulp hyperplasia, and histologic lesions and macrophage kinetics, but had slightly higher pulmonary bacterial burdens at the greatest measured time point. Together these findings indicate that C. burnetii primes but avoids cytosolic detection by NLRP3 inflammasomes, which are not required for the clinical resistance of C57BL/6 mice. Determining mechanisms employed by C. burnetii to avoid cytosolic detection via NLRP3 inflammasomes will be beneficial to the development of preventative and interventional therapies for Q fever.

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Apoptosis reprogramming triggered by splicing inhibitors sensitizes multiple myeloma cells to Venetoclax treatment

Haematologica ◽

10.3324/haematol.2021.279276 ◽

2021 ◽

Author(s):

Debora Soncini ◽

Claudia Martinuzzi ◽

Pamela Becherini ◽

Elisa Gelli ◽

Samantha Ruberti ◽

...

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Myeloid Cell ◽

Growth And Survival ◽

Bcl2 Family ◽

Cell Growth And Viability ◽

Novel Therapeutic Strategies ◽

The Impact

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells (PCs) from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant PCs. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of antiapoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for “priming” MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.

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166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab166 ◽

2013 ◽

Vol 25 (1) ◽

pp. 231

Author(s):

A. Alsaleh ◽

J. L. Pellerin ◽

C. Roux ◽

M. Larrat ◽

G. Chatagnon ◽

...

Keyword(s):

Embryo Transfer ◽

Coxiella Burnetii ◽

Q Fever ◽

Early Embryo ◽

Embryonic Cells ◽

Bovine Embryos ◽

Tissue Samples ◽

Worldwide Distribution

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Detection of significant bacterial loads in flushing media and tissue samples (oviducts and uterine horns) from the genital tracts of nonpregnant goats is a risk factor for in utero infection and transmission during embryo transfer (Alsaleh et al. 2011 CIMID 34, 355–360). The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the washing protocol recommend by the IETS for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and PCR, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9–9, 11–11, and 4–4 in replicates 1, 2, and 3, respectively) were placed in 1 mL of MEM containing 107 C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37°C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3–3, 5–5, and 2–2 in replicates 1, 2, and 3, respectively) were submitted to the same procedures but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 h at 13 000g. The washed embryos and pellets were tested by PCR. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first 5 washing fluids for ZP-free embryos and in the first 8 washing fluids for ZP-intact embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly demonstrate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to cling to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

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The phosphatidyl-myo-inositol dimannoside acyltransferase PatA is essential for Mycobacterium tuberculosis growth in vitro and in vivo

Journal of Bacteriology ◽

10.1128/jb.00439-20 ◽

2021 ◽

Author(s):

Francesca Boldrin ◽

Itxaso Anso ◽

Sogol Alebouyeh ◽

Iker A. Sevilla ◽

Mariví Geijo ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Biosynthetic Pathway ◽

Antimicrobial Agents ◽

Cell Envelope ◽

Etiologic Agent ◽

Permeability Barrier ◽

Macrophage Infection

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane associated acyltransferase that transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We validate that the function of PatA is vital for M. tuberculosis in vitro and in vivo. We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significant reduced levels of Ac1PIM2, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo. Altogether, the experimental data highlights the significance of the early steps of PIM biosynthetic pathway for M. tuberculosis physiology and uncover that PatA is a novel target for drug discovery programs against this major human pathogen. IMPORTANCE TB is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need of identifying new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis. These results highlight the importance of early steps of PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.

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Mitogenic and protective activity associated with a lipopolysaccharide from Coxiella burnetii

Canadian Journal of Microbiology ◽

10.1139/m78-259 ◽

1978 ◽

Vol 24 (12) ◽

pp. 1616-1618 ◽

Cited By ~ 3

Author(s):

Andrew Paquet Jr. ◽

Eppie D. Rael ◽

David Klassen ◽

Irene Martinez ◽

Oswald G. Baca

Keyword(s):

Candida Albicans ◽

Coxiella Burnetii ◽

Q Fever ◽

Specific Resistance ◽

Phase 1 ◽

Biological Properties ◽

Protective Activity ◽

Bacterial Endotoxins

A purified lipopolysaccharide (LPS) isolated from the rickettsial agent of Q fever, Coxiella burnetii phase 1, possesses some of the in vivo and in vitro biological properties previously only associated with bacterial endotoxins. The Coxiella LPS is mitogenic for guinea pig leukocytes, induces non-specific resistance in mice to virulent Candida albicans, causes dermal Schwartzman reactions, and is positive by the limulus lysate assay.

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Use of Supplemented or Human Material to Simulate PD Behavior of Antibiotics at the Target Site In Vitro

Pharmaceutics ◽

10.3390/pharmaceutics12080773 ◽

2020 ◽

Vol 12 (8) ◽

pp. 773

Author(s):

Alina Nussbaumer-Pröll ◽

Markus Zeitlinger

Keyword(s):

Antimicrobial Agents ◽

Biological Fluid ◽

In Vitro Models ◽

Body Fluids ◽

Growth Media ◽

Bacterial Strains ◽

Bacterial Killing ◽

The Impact

In antimicrobial drug development, in vitro antibiotic susceptibility testing is conducted in standard growth media, such as Mueller–Hinton broth (MHB). These growth media provide optimal bacterial growth, but do not consider certain host factors that would be necessary to mimic the in vivo bacterial environment in the human body. The present review aimed to include relevant data published between 1986 and 2019. A database search (PubMed) was done with text keywords, such as “MIC” (minimal inhibitory concentration), “TKC” (time kill curve), “blood”, “body fluid”, “PD” (pharmacodynamic), and “in vitro”, and 53 papers were ultimately selected. Additionally, a literature search for physiologic characteristics of body fluids was conducted. This review gives an excerpt of the complexity of human compartments with their physiologic composition. Furthermore, we present an update of currently available in vitro models operated either with adapted growth media or body fluids themselves. Moreover, the feasibility of testing the activity of antimicrobials in such settings is discussed, and pro and cons for standard practice methods are given. The impact on bacterial killing varies between individual adapted microbiological media, as well as direct pharmacodynamic simulations in body fluids, between bacterial strains, antimicrobial agents, and the compositions of the adjuvants or the biological fluid itself.

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