Identification of novel targets of azithromycin activity against Pseudomonas aeruginosa grown in physiologically relevant media

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.

Simulated Sunlight Decreases the Viability of SARS-CoV-2 in Mucus

This study examined the effect of simulated sunlight on the viability of SARS-CoV-2 spiked into tissue culture medium or mucus. The study revealed that inactivation took 37 minutes in medium and 107 minutes in mucus. These times-to-inactivation were unexpected since they are longer than have been observed in other studies.

Characterizing the response of Acinetobacter baumannii ATCC 17978 to azithromycin in multiple in vitro growth conditions

Multi-drug resistant (MDR) Acinetobacter baumannii is one of the most concerning pathogens in hospital infections. A. baumannii is categorized as an urgent threat by the U.S. Centers for Disease Control and the highest priority pathogen by the World Health Organization due to its propensity for broad antibiotic resistance and its associated high mortality rates. New treatment options are urgently needed for MDR A. baumannii infections. Our prior studies have demonstrated an unappreciated utility of the macrolide azithromycin (AZM) against MDR A. baumannii in tissue-culture medium. This finding is all the more surprising since AZM has no appreciable activity against A. baumannii in standard bacteriological media. The basis for this media-dependent activity of AZM against A. baumannii is not fully defined. In this study, we utilize a variety of techniques (growth dynamics, bacterial cytological profiling, RNA sequencing, and LC/MS) to profile the response of MDR A. baumannii to AZM in both standard bacteriological and more physiological relevant mammalian tissue-culture medium.

30 Supplementation of invitro culture medium with linoleic acid albumin improves bovine embryo survivability in low-temperature storage at 4°C

The objective of this study was to investigate the addition of linoleic acid albumin (LAA) to invitro culture medium to improve the development of blastocysts with low permeability and fluidity in serum-supplemented medium. Currently, vitrification and slow freezing are performed as cryopreservation methods of bovine embryos. Recently, it was reported that invivo embryos stored at 4°C for 168h retained their pregnancy competence. For IVF, embryo development and cryotolerance are highly influenced by the presence of serum in the culture medium. Thus, this study aimed to confirm the survivability of invitro fertilized embryos cultured in medium supplemented with LAA after low-temperature storage. The cumulus-oocyte complex collected from slaughterhouse-derived ovaries was matured for 20h in tissue culture medium-199 supplemented with 0.02 AUmL−1 FSH and 5% calf serum (CS). After cumulus-oocyte complexes were incubated with 5×106 spermatozoa per mL for 6h and the presumptive zygotes were removed, their cumulus cells were cultured with CR1aa supplemented with 5% CS (control) or CR1aa supplemented with 0.3mgmL−1 LAA and 5% CS (LAA group) for 7 or 8 days. The code 1 or 2 blastocysts that developed on Days 7 and 8 were used in this study. The developed blastocysts were equilibrated to the low temperature storage medium (25mM HEPES buffered tissue culture medium-199 supplemented with 50% fetal calf serum (FCS)) for 20min and stored in a refrigerator at 4±1°C for 48 or 72h. After low-temperature storage, the blastocysts were diluted in a buffer (Dulbecco's phosphate-buffered saline) supplemented with 10% FCS for 10min, washed three times with tissue culture medium-199 supplemented with 20% FCS and 0.1mM β-mercaptoethanol, and cultured in the same medium. Thereafter, blastocyst survival (re-expansion) and hatching were observed for up to 72h. The data were analysed using the chi-squared test with Yates's correction. The results of 48-h low-temperature storage of blastocysts for the control (n=55) and LAA (n=28) groups showed significant differences (P<0.01) in survival (41.8 and 89.3%, respectively) and hatched rates (20.0 and 89.3%, respectively). The results of 72-h low-temperature storage of blastocysts for the control (n=13) and LAA (n=38) groups showed significant differences (P<0.01) in survival (7.7 and 52.6%, respectively) and hatched rates (7.7 and 52.6%, respectively). These results suggested that supplementation of culture media with LAA improved the survivability of expanded blastocysts after low-temperature storage.