Considerations for Dose Selection and Clinical Pharmacokinetics/Pharmacodynamics for the Development of Antibacterial Agents.

In June 2017, The National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, organized a workshop entitled "Pharmacokinetics-Pharmacodynamics (PK/PD) for Development of Therapeutics against Bacterial Pathogens" to discuss details and critical parameters of various PK/PD methods and identify approaches for linking human pharmacokinetic (PK) data and drug efficacy analyses. The workshop participants included individuals from academia, industry, and government. This and the accompanying minireview on nonclinical PK/PD summarize the workshop discussions and recommendations. It is important to consider how information like PK/PD can support the clinical effectiveness of new antibacterial drugs, as PK/PD data have become central to antibacterial drug development programs. Key clinical considerations for antibacterial dose selection and clinical PK/PD characterization discussed in this minireview include a robust assessment of PK in the patient population of interest, critical considerations for assessing drug penetration in the lung for the treatment of pneumonia, and an emphasis on special populations, including patients with renal impairment and augmented renal function, as well as on dosing in obese and pediatric patients. Successful application of such approaches is now used to provide a more informative drug development package to support the approval of new antibiotics.

Haematological, biochemical and bone density parameters in vegetarians and non-vegetarians / Parámetros de densidad ósea, hematológica, y bioquímica en vegetarianos y no vegetarianos

OBJECTIVE: The objective is to determine any possible differences between haematological, biochemical and bone mineral density in vegetarians (vegans and lacto-ovovegetarians) and non-vegeterians. METHODS: The examined group consisted of 100 individuals: 50 non-vegetarians and 50 vegetarians. The vegetarian group was further divided in 2 subgroups: 20 vegans and 30 lacto-ovovegetarians. In all participants, plasma levels of erythrocytes, haemoglobin, haematocrit, iron, low density lipoprotein, (LDL), high density lipoprotein (HDL) total cholesterol, triglycerides and glucose were measured. Quantitative ultrasound parameters of the right calcaneus were determined in all participants. RESULTS: The results showed that lacto-ovovegetarians had statistically significantly higher red blood cell counts and haematocrit values than non-vegetarians. Vegans also had higher haematocrit values than non-vegetarians. Statistically significant differences were found between iron plasma levels in the examined groups. Iron levels were lower in non-vegetarians than in vegans and lacto-ovovegetarians. Non-vegetarians had much higher levels of cholesterol, triglycerides and LDL than the other two groups, but there were no differences found between same values in vegans and lacto-ovovegetarians. CONCLUSION: A well planned and balanced vegetarian diet, with avoidance of risk factors, does not result in abnormalities in laboratory tests and bone status parameters.

OBJETIVO: El objetivo es determinar las diferencias entre la densidad hematológica, bioquímica y mineral ósea en vegetarianos (veganos y ovolactovegetarianos) y no vegetarianos. MÉTODOS: El grupo examinado consistía en 100 individuos: 50 no vegetarianos y 50 vegetarianos. El grupo vegetariano estaba a su vez dividido en 2 subgrupos: 20 veganos y 30 ovolactovegetarianos. A todos los participantes se les midieron los niveles plasmáticos de eritrocitos, hemoglobina, hematocrito, hierro, lipoproteína de baja densidad (LDL), lipoproteína de alta densidad (HDL), colesterol total, triglicéridos, y glucosa. Se determinaron los parámetros cuantitativos de ultrasonidos del calcáneo derecho en todos los participantes. RESULTADOS: Los resultados mostraron que los ovolactovegetarianos tuvieron conteos de glóbulos rojos y valores de hematocrito significativamente más altos en términos estadísticos, que los no vegetarianos. Los veganos también tuvieron valores de hematocrito más altos que los no vegetarianos. Se halló diferencia estadísticamente significativa entre los niveles séricos de hierro en los grupos examinados. Los niveles de hierro fueron más bajos en los no vegetarianos que en los veganos y los ovolacto-vegetarianos. Los no vegetarianos tuvieron niveles de colesterol, triglicéridos y LDL mucho más altos que los otros dos grupos, pero no se encontraron diferencias entre los mismos valores en los veganos y los ovolactovegetarianos. CONCLUSIÓN: Podemos concluir que una dieta vegetariana bien planeada y equilibrada, que evite factores de riesgo, no trae consigo anormalidades en las pruebas de laboratorio y los parámetros del estado óseo.

Haematological, biochemical and bone density parameters in vegetarians and non-vegetarians.

OBJECTIVE: The objective is to determine any possible differences between haematological, biochemical and bone mineral density in vegetarians (vegans and lacto-ovovegetarians) and non-vegeterians. METHODS: The examined group consisted of 100 individuals: 50 non-vegetarians and 50 vegetarians. The vegetarian group was further divided in 2 subgroups: 20 vegans and 30 lacto-ovovegetarians. In all participants, plasma levels of erythrocytes, haemoglobin, haematocrit, iron, low density lipoprotein, (LDL), high density lipoprotein (HDL) total cholesterol, triglycerides and glucose were measured. Quantitative ultrasound parameters of the right calcaneus were determined in all participants. RESULTS: The results showed that lacto-ovovegetarians had statistically significantly higher red blood cell counts and haematocrit values than non-vegetarians. Vegans also had higher haematocrit values than non-vegetarians. Statistically significant differences were found between iron plasma levels in the examined groups. Iron levels were lower in non-vegetarians than in vegans and lacto-ovovegetarians. Non-vegetarians had much higher levels of cholesterol, triglycerides and LDL than the other two groups, but there were no differences found between same values in vegans and lacto-ovovegetarians. CONCLUSION: A well planned and balanced vegetarian diet, with avoidance of risk factors, does not result in abnormalities in laboratory tests and bone status parameters.

Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity.

Resistance to innate immunity is essential for salmonellae pathogenesis. The salmonellae PhoP/PhoQ regulators sense host environments to promote remodeling of the bacterial envelope. This remodeling includes enzymes that modify lipopolysaccharide (LPS). Modified LPS promotes bacterial survival by increasing resistance to cationic antimicrobial peptides and by altered host recognition of LPS.

Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria.

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His(6)-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of Yersinia and Bordetella species. PagP may provide an adaptive response toward both Mg(2+) limitation and host innate immune defenses.

A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides.

The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (alpha-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue alpha-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to alpha-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.

How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses.

Bacterial pathogens regulate the expression of virulence factors in response to environmental signals. In the case of salmonellae, many virulence factors are regulated via PhoP/PhoQ, a two-component signal transduction system that is repressed by magnesium and calcium in vitro. PhoP/PhoQ-activated genes promote intracellular survival within macrophages, whereas PhoP-repressed genes promote entrance into epithelial cells and macrophages by macropinocytosis and stimulate epithelial cell cytokine production. PhoP-activated genes include those that alter the cell envelope through structural alterations of lipopolysaccharide and lipid A, the bioactive component of lipopolysaccharide. PhoP-activated changes in the bacterial envelope likely promote intracellular survival by increasing resistance to host cationic antimicrobial peptides and decreasing host cell cytokine production.

Sequence and phylogenetic analysis of the Borrelia burgdorferi secA gene.

A Borrelia burgdorferi secA homologue was cloned and the complete DNA sequence was determined. The deduced protein sequence consists of 899 amino acids and shows a high degree of homology to SecA homologues from other Bacteria and photosynthetic plastids. The presence of the secA gene in Spirochetes suggests that this gene is present in most if not all major lineages within Bacteria. The ease of isolation of secA by conservation of its ATP-binding motifs combined with its extreme conservation in protein secretion pathways and the presence of a phylogenetic sequence marker in one of its ATP-binding domains makes this gene useful for phylogenetic analysis of Bacteria and photosynthetic plastids.

Cloning and analysis of a Borrelia burgdorferi membrane-interactive protein exhibiting haemolytic activity.

We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted alpha-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane-associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X-114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi.