The origins of haplotype 58 (H58) Salmonella enterica serovar Typhi.

Antimicrobial resistance (AMR) poses a serious threat to the clinical management of typhoid fever. AMR in Salmonella Typhi (S. Typhi) is commonly associated with the H58 lineage, a lineage that arose comparatively recently before becoming globally disseminated. To better understand when and how H58 emerged and became dominant, we performed detailed phylogenetic analyses on contemporary genome sequences from S. Typhi isolated in the period spanning the emergence. Our dataset, which contains the earliest described H58 S. Typhi organism, indicates that ancestral H58 organisms were already multi-drug resistant (MDR). These organisms emerged spontaneously in India in 1987 and became radially distributed throughout South Asia and then globally in the ensuing years. These early organisms were associated with a single long branch, possessing mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent use of fluoroquinolones led to several independent mutations in gyrA. The ability of H58 to acquire and maintain AMR genes continues to pose a threat, as extensively drug-resistant (XDR; MDR plus resistance to ciprofloxacin and third generation cephalosporins) variants, have emerged recently in this lineage. Understanding where and how H58 S. Typhi originated and became successful is key to understand how AMR drives successful lineages of bacterial pathogens. Additionally, these data can inform optimal targeting of typhoid conjugate vaccines (TCVs) for reducing the potential for emergence and the impact of new drug-resistant variants. Emphasis should also be placed upon the prospective identification and treatment of chronic carriers to prevent the emergence of new drug resistant variants with the ability to spread efficiently.

Back-pocket optimization of 2-aminopyrimidine-based macrocycles leads to potent dual EPHA2/GAK kinase inhibitors with antiviral activity.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.

Back-Pocket Optimization of 2-Aminopyrimidine-Based Macrocycles Leads to Potent EPHA2/GAK Kinase Inhibitors.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here we have optimized 2-aminopyrimidine-based macrocycles to use these compounds as chemical tools for the ephrin kinase family. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55, a potent inhibitor of EPHA2/A4 and GAK. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/A4 and GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells. However, further investigations are needed to determine whether this was a compound-specific effect or target-related.

PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2.

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

The repurposing of Tebipenem pivoxil as alternative therapy for severe gastrointestinal infections caused by extensively drug-resistant Shigella spp.

Background: Diarrhoea remains one of the leading causes of childhood mortality globally. Recent epidemiological studies conducted in low-middle income countries (LMICs) identified Shigella spp. as the first and second most predominant agent of dysentery and moderate diarrhoea, respectively. Antimicrobial therapy is often necessary for Shigella infections; however, we are reaching a crisis point with efficacious antimicrobials. The rapid emergence of resistance against existing antimicrobials in Shigella spp. poses a serious global health problem. Methods: Aiming to identify alternative antimicrobial chemicals with activity against antimicrobial resistant Shigella, we initiated a collaborative academia-industry drug discovery project, applying high-throughput phenotypic screening across broad chemical diversity and followed a lead compound through in vitro and in vivo characterisation. Results: We identified several known antimicrobial compound classes with antibacterial activity against Shigella. These compounds included the oral carbapenem Tebipenem, which was found to be highly potent against broadly susceptible Shigella and contemporary MDR variants for which we perform detailed pre-clinical testing. Additional in vitro screening demonstrated that Tebipenem had activity against a wide range of other non-Shigella enteric bacteria. Cognisant of the risk for the development of resistance against monotherapy, we identified synergistic behaviour of two different drug combinations incorporating Tebipenem. We found the orally bioavailable prodrug (Tebipenem pivoxil) had ideal pharmacokinetic properties for treating enteric pathogens and was effective in clearing the gut of infecting organisms when administered to Shigella-infected mice and gnotobiotic piglets. Conclusions: Our data highlight the emerging antimicrobial resistance crisis and shows that Tebipenem pivoxil (licenced for paediatric respiratory tract infections in Japan) should be accelerated into human trials and could be repurposed as an effective treatment for severe diarrhoea caused by MDR Shigella and other enteric pathogens in LMICs. Funding: Tres Cantos Open Lab Foundation (projects TC239 and TC246), the Bill and Melinda Gates Foundation (grant OPP1172483) and Wellcome (215515/Z/19/Z).

A Phylogenetic and Phenotypic Analysis of Salmonella enterica Serovar Weltevreden, an Emerging Agent of Diarrheal Disease in Tropical Regions.

Salmonella enterica serovar Weltevreden (S. Weltevreden) is an emerging cause of diarrheal and invasive disease in humans residing in tropical regions. Despite the regional and international emergence of this Salmonella serovar, relatively little is known about its genetic diversity, genomics or virulence potential in model systems. Here we used whole genome sequencing and bioinformatics analyses to define the phylogenetic structure of a diverse global selection of S. Weltevreden. Phylogenetic analysis of more than 100 isolates demonstrated that the population of S. Weltevreden can be segregated into two main phylogenetic clusters, one associated predominantly with continental Southeast Asia and the other more internationally dispersed. Subcluster analysis suggested the local evolution of S. Weltevreden within specific geographical regions. Four of the isolates were sequenced using long read sequencing to produce high quality reference genomes. Phenotypic analysis in Hep-2 cells and in a murine infection model indicated that S. Weltevreden were significantly attenuated in these models compared to the classical S. Typhimurium reference strain SL1344. Our work outlines novel insights into this important emerging pathogen and provides a baseline understanding for future research studies.

The transfer and decay of maternal antibody against Shigella sonnei in a longitudinal cohort of Vietnamese infants.

BACKGROUND: Shigella sonnei is an emergent and major diarrheal pathogen for which there is currently no vaccine. We aimed to quantify duration of maternal antibody against S. sonnei and investigate transplacental IgG transfer in a birth cohort in southern Vietnam. METHODS AND RESULTS: Over 500-paired maternal/infant plasma samples were evaluated for presence of anti-S. sonnei-O IgG and IgM. Longitudinal plasma samples allowed for the estimation of the median half-life of maternal anti-S. sonnei-O IgG, which was 43 days (95% confidence interval: 41-45 days). Additionally, half of infants lacked a detectable titer by 19 weeks of age. Lower cord titers were associated with greater increases in S. sonnei IgG over the first year of life, and the incidence of S. sonnei seroconversion was estimated to be 4/100 infant years. Maternal IgG titer, the ratio of antibody transfer, the season of birth and gestational age were significantly associated with cord titer. CONCLUSIONS: Maternal anti-S. sonnei-O IgG is efficiently transferred across the placenta and anti-S. sonnei-O maternal IgG declines rapidly after birth and is undetectable after 5 months in the majority of children. Preterm neonates and children born to mothers with low IgG titers have lower cord titers and therefore may be at greater risk of seroconversion in infancy.

A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae.

Multidrug-resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource-poor hospital settings. Through whole-genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high-dependency wards in a hospital in Kathmandu during 2012 with a case-fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage-specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward-specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM-1 containing lineage in 2011 would have alerted the high-dependency ward staff to intervene. We argue that some form of real-time genetic characterisation, alongside clade-specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high- and low-income settings.