Microbiotix, Inc.

Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.

Efflux pump inhibitors (EPIs) offer an attractive therapeutic option when combined with existing classes. However, their optimal dosing strategies are unknown.

MICs of ciprofloxacin (CIP)+/−chlorpromazine, phenylalanine-arginine β naphthylamide (PAβN) and a developmental molecule MBX-4191 were determined and the pharmacodynamics (PD) was studied in an in vitro model employing Escherichia coli MG1655 and its isogenic MarR mutant (I1147). Exposure ranging experiments were performed initially then fractionation. Changes in bacterial load and population profiles were assessed. Strains recovered after EPI simulations were studied by WGS.

The CIPMICs for E. coli MG1655 and I1147 were 0.08 and 0.03 mg/L. Chlorpromazine at a concentration of 60 mg/L, PAβN concentrations of 30 mg/L and MBX-4191 concentrations of 0.5–1.0 mg/L reduced CIP MICs for I1147 and enhanced bacterial killing. Using CIP at an AUC of 1.2 mg·h/L, chlorpromazine AUC was best related to reduction in bacterial load at 24 h, however, when the time drug concentration was greater than 25 mg/L (T > 25 mg/L) chlorpromazine was also strongly related to the effect. For PaβN with CIP AUC, 0.6 mg·h/L PaβN AUC was best related to a reduction in bacterial load. MBX-4191T > 0.5–0.75 mg·h/L was best related to reduction in bacterial load. Changes in population profiles were not seen in experiments of ciprofloxacin + EPIs. WGS of recovered strains from simulations with all three EPIs showed mutations in gyrA, gyrB or marR.

AUC was the pharmacodynamic driver for chlorpromazine and PAβN while T > threshold was the driver for MBX-4191 and important in the activity of chlorpromazine and PAβN. Changes in population profiles did not occur with combinations of ciprofloxacin + EPIs, however, mutations in gyrA, gyrB and marR were detected.

Effective surveillance for epidemic-prone viral diseases is essential for emergency preparedness to respond to threats and occurrences of pandemics. While it is difficult and expensive to conduct health facility-based surveillance, there is a growing interest in conducting sewage-based epidemiological studies to monitor the health of the urban population because of the relative ease of sample collection and the availability of advanced molecular techniques for the detection of pathogens in the sewage. Sewage samples offer unique means to study the aggregate health of the population as opposed to the monitoring of the health of any individual by traditional methods. We worked together with the Ministry of Public Works in Kuwait and developed a platform for the collection and testing of sewage samples from different regions of Kuwait for studying population health. In this report, we describe the results of a cross-sectional study conducted between 16 and 23 September 2019 in an attempt to detect influenza, Noro, Rota, hepatitis A, and hepatitis E viruses in urban sewage samples collected in Kuwait. All five targeted viruses were detected in the samples collected from urban wastewater in Kuwait using reverse-transcriptase quantitative PCR (RT-qPCR). We recently checked for the presence of SARS-CoV-2 in the stored cDNA samples and confirmed the absence of SARS-CoV-2 in them. This is the first report that demonstrates the preparedness in Kuwait for using sewage samples for the detection and monitoring of many pathogenic viruses which may greatly increase the capacity of the country to deal with a viral disease outbreak in the future.