In vitro activity of tigecycline and comparators against carbapenem-susceptible and resistant Acinetobacter baumannii clinical isolates in Italy

Background

Effective neutralization of active agents is essential to obtain valid efficacy results, especially when non-volatile active agents like chlorhexidine digluconate (CHG) are tested. The aim of this study was to determine an effective and non-toxic neutralizing mixture for a propan-1-ol solution containing 2% CHG.

Methods

Experiments were carried out according to ASTM E 1054-02. The neutralization capacity was tested separately with five challenge microorganisms in suspension, and with a rayon swab carrier. Either 0.5 mL of the antiseptic solution (suspension test) or a saturated swab with the antiseptic solution (carrier test) was added to tryptic soy broth containing neutralizing agents. After the samples were mixed, aliquots were spread immediately and after 3 h of storage at 2 – 8°C onto tryptic soy agar containing a neutralizing mixture.

Results

The neutralizer was, however, not consistently effective in the suspension test. Immediate spread yielded a valid neutralization with Staphylococcus aureus, Staphylococcus epidermidis and Corynebacterium jeikeium but not with Micrococcus luteus (p < 0.001) and Candida albicans (p < 0.001). A 3-h storage period of the neutralized active agents in suspension resulted in significant carry-over activity of CHG in addition against Staphylococcus epidermidis (p < 0.001) and Corynebacterium jeikeium (p = 0.044). In the carrier test, the neutralizing mixture was found to be effective and non toxic to all challenge microorganisms when spread immediately. However, after 3 h storage of the neutralized active agents significant carry-over activity of CHG against Micrococcus luteus (p = 0.004; Tukey HSD) was observed.

Conclusion

Without effective neutralization in the sampling fluid, non-volatile active ingredients will continue to reduce the number of surviving microorganisms after antiseptic treatment even if the sampling fluid is kept cold straight after testing. This can result in false-positive antiseptic efficacy data. Attention should be paid during the neutralization validation process to the amount of antiseptic solution, the storage time and to the choice of appropriate and sensitive microorganisms.     

PhosphoScan: a probability-based method for phosphorylation site prediction using MS2/MS3 pair information

Phosphopeptide identification and phosphorylation site localization are crucial aspects of many biological studies. Furthermore, multiple phosphorylations of peptides make site localization even more difficult. We developed a probability-based method to unambiguously determine phosphorylation sites within phosphopeptides using MS2/3 pair information. A comparison test was performed with SEQUEST and MASCOT predictions using a spectral data set from a synthetic doubly phosphorylated peptide, and the results showed that PhosphoScan analysis yielded a 63% phosphopeptide localization improvement compared with SEQUEST and a 57% improvement compared with MASCOT.     

Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain

The HypF N-terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3-5 nm and 7-9 nm in width, whereas fibrils of 7-9 nm and 12-20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.     

Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.     

Aspartic-129 is an essential residue in the catalytic mechanism of the low Mr phosphotyrosine protein phosphatase

The crystal structure of the bovine liver low M_r phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by ¹H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k₂) and dephosphorylation (k₃) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.     

Oxidative Stress in the Heart of Rats Infected with Trypanosoma evansi

This study was conducted to investigate the occurrence of oxidative stress in the heart tissue of rats infected with Trypanosoma evansi. Rats were divided into 2 groups (A and B) with 12 animals each, and further subdivided into 4 subgroups (A1 and A2, 6 animals/each; and B1 and B2, 6 animals/each). Animals in the groups B1 and B2 were subcutaneously inoculated with T. evansi. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD), glutathione S-transferase activity (GST), reduced glutathione activity (GSH), and non-protein thiols (NPSH) in the heart tissue were evaluated. At day 5 and 15 post-infection (PI), an increase in the TBARS levels and a decrease in the SOD activity (P<0.05) were observed. GSH and GST activities were decreased in infected animals at day 15 PI (P<0.05). Considering the proper functioning of the heart, it is possible that the changes in the activity of these enzymes involved in the oxidative stress may be related, at least in part, in the pathophysiology of rats infected with T. evansi.     

Paternal exposure to excessive methionine altered behavior and neurochemical activities in zebrafish offspring

An increase in plasma l-methionine (Met) levels, even if transitory, can cause important toxicological alterations in the affected individuals. Met is essential in the regulation of epigenetic mechanisms and its influence on the subsequent generation has been investigated. However, few studies have explored the influence of a temporary increase in Met levels in parents on their offspring. This study evaluated the behavioral and neurochemical effects of parental exposure to high Met concentration (3 mM) in zebrafish offspring. Adult zebrafish were exposed to Met for 7 days, maintained for additional 7 days in tanks that contained only water, and then used for breeding. The offspring obtained from these fish (F1) were tested in this study. During the early stages of offspring development, morphology, heart rate, survival, locomotion, and anxiety-like behavior were assessed. When these animals reached the adult stage, locomotion, anxiety, aggression, social interaction, memory, oxidative stress, and levels of amino acids and neurotransmitters were analyzed. F1 larvae Met group presented an increase in the distance and mean speed when compared to the control group. F1 adult Met group showed decreased anxiety-like behavior and locomotion. An increase in reactive oxygen species was also observed in the F1 adult Met group whereas lipid peroxidation and antioxidant enzymes did not change when compared to the control group. Dopamine, serotonin, glutamate, and glutathione levels were increased in the F1 adult Met group. Taken together, our data show that even a transient increase in Met in parents can cause behavioral and neurochemical changes in the offspring, promoting transgenerational effects.

     

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.