Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens

Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials

Evaluation of epidemiological cut-off values indicates that biocide resistant subpopulations are uncommon in natural isolates of clinically-relevant microorganisms

To date there are no clear criteria to determine whether a microbe is susceptible to biocides or not. As a starting point for distinguishing between wild-type and resistant organisms, we set out to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) distributions for four common biocides; triclosan, benzalkonium chloride, chlorhexidine and sodium hypochlorite for 3319 clinical isolates, with a particular focus on Staphylococcus aureus (N = 1635) and Salmonella spp. (N = 901) but also including Escherichia coli (N = 368), Candida albicans (N = 200), Klebsiella pneumoniae (N = 60), Enterobacter spp. (N = 54), Enterococcus faecium (N = 53), and Enterococcus faecalis (N = 56). From these data epidemiological cut-off values (ECOFFs) are proposed. As would be expected, MBCs were higher than MICs for all biocides. In most cases both values followed a normal distribution. Bimodal distributions, indicating the existence of biocide resistant subpopulations were observed for Enterobacter chlorhexidine susceptibility (both MICs and MBCs) and the susceptibility to triclosan of Enterobacter (MBC), E. coli (MBC and MIC) and S. aureus (MBC and MIC). There is a concern on the potential selection of antibiotic resistance by biocides. Our results indicate however that resistance to biocides and, hence any potential association with antibiotic resistance, is uncommon in natural populations of clinically relevant microorganisms. \ua9 2014 Morrissey et al

A Functional Genomics Approach to Establish the Complement of Carbohydrate Transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium∶solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection

Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia : original images

Original Image files for the figures of the paper "Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia

Furi JB00370-19 Supplementary data.pdf

Supplementary data for JB00370-19 Furi et al., Methylation warfare: interaction of pneumococcal bacteriophages with their host. submitted to Journal of Bacteriology</div

Zamudio et al Environmental Microbiology 2020 SUPPELMENTARY DATA 10.1111/1462-2920.15111

Zamudio R, RD Haigh, JD Ralph, M De Ste Croix, T Tasara, K Zurfluh, MJ Kwun, AD Millard, SD Bentley, NJ Croucher, R Stephan, MR Oggioni. 2020. Lineage specific evolution and gene flow in Listeria monocytogenes is independent of bacteriophages. Environmental Microbiology. 2020 doi 10.1111/1462-2920.15111 Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracing of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage specific characteristics call for a more detailed understanding of evolution. In our work we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.</p

Interaction of Klebsiella pneumoniae with tissue macrophages in a murine infection model and ex-vivo porcine organ perfusions: an exploratory investigation. 2021 Lancet Microbe - Original Images and Data Files

ORIGINAL IMAGE FILESAbstract Background: Hypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K. pneumoniae strains (non-hvKp), including lineages which have acquired carbapenem resistance (CRKp), do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanisms underpinning abscess formation in hvKp infections. Methods: To study the pathophysiology of abscess formation, mice were infected intravenously with hvKp and non-hvKp isolates and intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy. Microbiological and microscopy analysis of an ex vivo model of porcine liver and spleen infection was used to confirm within-macrophage replication. Findings: We demonstrate that hvKp resisted phagocyte-mediated clearance and replicated in murine liver macrophages to form already 8 hours post challenge clusters with an average of 7.0 bacteria per cell (SD 6.1), whilst non-hvKp were efficiently cleared. hvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation of as early as 24h post-infection. Experiments in porcine organs, which share a high functional and anatomical resemblance to human organs, provided strong evidence for the prospensity of hvKp to replicate within the hepatic macrophages. Intepretation: These findings demonstrate subversion of innate immune processes in the liver by Kp and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses.</div

Pneumococcal Surface Protein C Contributes to Sepsis Caused by Streptococcus pneumoniae in Mice

The role of pneumococcal surface protein C (PspC; also called SpsA, CbpA, and Hic) in sepsis by Streptococcus pneumoniae was investigated in a murine infection model. The pspC gene was deleted in strains D39 (type 2) and A66 (type 3), and the mutants were tested by being injected intravenously into mice. The animals infected with the mutant strains showed a significant increase in survival, with the 50% lethal dose up to 250-fold higher than that for the wild type. Our findings indicate that PspC affords a decisive contribution to sepsis development