Computational identification of a systemic antibiotic for gram-negative bacteria.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its ß-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.

The Influence of the Toxin/Antitoxin mazEF on Growth and Survival of Listeria monocytogenes under Stress.

A major factor in the resilience of Listeria monocytogenes is the alternative sigma factor B (σB). Type II Toxin/Antitoxin (TA) systems are also known to have a role in the bacterial stress response upon activation via the ClpP or Lon proteases. Directly upstream of the σB operon in L. monocytogenes is the TA system mazEF, which can cleave mRNA at UACMU sites. In this study, we showed that the mazEF TA locus does not affect the level of persister formation during treatment with antibiotics in lethal doses, but exerts different effects according to the sub-inhibitory stress added. Growth of a ΔmazEF mutant was enhanced relative to the wildtype in the presence of sub-inhibitory norfloxacin and at 42 °C, but was decreased when challenged with ampicillin and gentamicin. In contrast to studies in Staphylococcus aureus, we found that the mazEF locus did not affect transcription of genes within the σB operon, but MazEF effected the expression of the σB-dependent genes opuCA and lmo0880, with a 0.22 and 0.05 fold change, respectively, compared to the wildtype under sub-inhibitory norfloxacin conditions. How exactly this system operates remains an open question, however, our data indicates it is not analogous to the system of S. aureus, suggesting a novel mode of action for MazEF in L. monocytogenes.

The Small Colony Variant of Listeria monocytogenes Is More Tolerant to Antibiotics and Has Altered Survival in RAW 264.7 Murine Macrophages.

Small Colony Variant (SCV) cells of bacteria are a slow-growing phenotype that result from specific defects in the electron transport chain. They form pinpoint colonies on agar plates and have a variety of phenotypic characteristics, such as altered carbon metabolism, decreased toxin and lytic enzyme production, aminoglycoside resistance, and increased intracellular persistence. They are clinically relevant in Staphylococcus aureus and Pseudomonas aeruginosa, serving as a reservoir for recurrent or prolonged infections. Here, we found that a SCV mutant in the foodborne pathogen Listeria monocytogenes (strain SCV E18), similar to the high persister mutant phenotype, survived significantly better than the wild type when exposed over a 48-h period to concentrations above Minimal Inhibitory Concentration for most tested antibiotics. SCV E18 survived more poorly than the wildtype in unactivated RAW264.7 macrophage cells, presumably because of its reduced listeriolysin O expression, however, it survived better in reactive oxygen species producing, phorbol 12-myristate 13-acetate-activated macrophages. Although SCV E18 was sensitive to oxygen as it entered the stationary phase, it was significantly more tolerant to H2O2 than the wild type, which may result from a shift in metabolism, however, further investigation is needed to resolve this. SCV E18 is a spontaneous mutant with a point mutation in the hemA gene. A wild type copy of hemA was complemented on plasmid pSOG30222, which restored the wild type phenotype. The results reported here suggest that the SCV of L. monocytogenes could be of clinical importance and highlight a need for adequate clinical screening for this phenotype, as it could affect antibiotic treatment outcomes.

An adaptation of the MMPI-2 Meyers Index for the MMPI-2-RF.

Using an overall sample of 278 individuals who had taken the Minnesota Multiphasic Personality Inventory-Second Edition (MMPI-2) and who had clear diagnostic information available in their medical records, the Meyers Index (MI) for the MMPI-2 (Meyers, Millis, & Volkert, 2002 ) was calculated for each individual, and a new version of the MI created for the MMPI-2 Restructured Form (MMPI-2-RF) was calculated. The MI is a method of combining multiple MMPI-2 validity scales into a single weighted index to assess exaggerated self-report on the MMPI-2. The new index is intended to provide the same type of global assessment of validity but for the MMPI-2-RF (MI-r). The MI and the MI-r were compared at both individual and group levels and were found to correlate well (r = .87). Diagnostic groups of litigants and nonlitigants of traumatic brain injury, chronic pain, and posttraumatic stress disorder were also examined; and the performance of the MI and the MI-r were similar. Similarly, the pass and fail agreement rate for the two scales was 93%. The results indicate that the MI and MI-r perform very similarly and are good methods of assessing overall validity of MMPI-2 and MMPI-2-RF test performance.

Are Self-Ratings of Functional Difficulties Objective or Subjective?

In this study, we compared objective neuropsychological data using the Meyers Neuropsychological Battery (MNB; Meyers & Rohling, 2004 ) and self-report measures of emotional distress using the Symptom Checklist 90-Revised (SCL-90-R; Derogatis, 1994 ) with self-ratings of functional difficulties as measured by the Patient Competency Rating Scale (PCRS; Prigatano, 1986 ). The results showed a high correlation between the PCRS and scales on the SCL-90-R (r = .65), whereas correlation with the overall test battery mean of the MNB was quite small (r = .18). Our results indicate that self-report of cognitive difficulties is more related to current emotional distress than to objective measures. Therefore, any diagnostic considerations that rely on self-report need to be tempered by considerations of current emotional status. This has implications for diagnoses such as posttraumatic stress disorder and other diagnoses that rely on self-report as a source of diagnostic information.