Antimicrobials and antimicrobial resistance (AAMR)

Antibiotics resistance is an emerging threat for the health systems worldwide.700000 deaths annually are attributed to antibiotic resistant pathogens. The predictions for 2050 are devastating. By 2050, 10 million deaths that are linked to antibiotic resistance are expected. The antibiotic resistance along with the shortage of new antibiotics have created an antibiotic crisis. One solution to antimicrobial resistance could be the investment in new innovative methods against antimicrobial resistance. In the present study, two research projects with innovative strategies were undertaken. The first project examined the route of transmission of Enterococcus faecium in a suspected outbreak using WGS. The second project concerned the characterisation of chemosensory proteins in the pathogen Pseudomonas aeruginosa in order to elucidate the mechanism by which the bacterium senses anti-attachment surface

SadB is a Pseudomonas aeruginosa global regulator

Bacteria can adhere to almost any surface and form resistant biofilm communities. These pose a major clinical burden as they can form on medical devices such as catheters and are hard to prevent. Catheter- associated urinary tract infections (CAUTIs) are the most common nosocomial infection as they represent ~40% of all nosocomial infections. Hook et al., previously developed a series of polymers that prevent biofilm formation. Following a screen of 20,000 polymers and copolymers and subsequent scale-up, the hit acrylate polymer, EGdPEA has been clinically approved for the coating of urinary tract catheters and presents superior antibiofilm properties compared with silver-embedded silicone catheters. The bacterial biofilm resistance mechanism and surface sensing pathways involved are under investigation. In P. aeruginosa surface sensing includes the flagella, type IV pili and the wsp system. The present study sought to investigate SadB, an essential gene for biofilm formation and CheA, a histidine kinase of the chemotaxis system for their involvement in surface sensing. SadB is involved in the switch from reversible to irreversible surface attachment. Expression of sadB under a constitutive promoter overcomes the resistance of EGdPEA to biofilm formation. CheA is responsible for transmitting environmental signals to the cell and activating Pch phosphodiesterase, which is important in surface sensing. Biofilm and motility assays and monitoring on-surface gene expression were used as a test of potential involvement in surface sensing. In agreement with the literature, SadB was found to be essential for biofilm formation and to affect swarming motility. sadB was also found to be differentially expressed on the anti and pro-biofilm surfaces. In contrast, CheA affected swarming but did not affect biofilm formation on most polymer surfaces and glass. The promoter of cheA also controls the cheI operon with 13 more genes. The expression of the operon was found to be different on different surfaces but CheA is unlikely to be important in surface sensing. The literature and findings of the present study highlighted the importance of sadB in surface sensing, but the regulatory pathway was still unknown. Whole transcriptome analysis (RNAseq) was employed to investigate the downstream regulation of sadB in an untargeted manner. RT-PCR and phenotypic assays were used to validate the data. SadB was found to be a global regulator controlling up to 50% of the genome. sadB was shown to negatively affect the pqs system and subsequently the rhl system, rhamnolipid and pyocyanin production and the denitrification pathway. The levels of the PQS signal, pyocyanin and rhamnolipids were also shown to be affected by sadB. The denitrification pathway downregulation in the absence of sadB was shown to affect growth under microaerophilic conditions. Furthermore, sadB positively affected biofilm related targets such as Psl and c-di-GMP production. Advances were made in the present study in discovering the downstream targets of sadB that explain some of the observed phenotypes, however the function of SadB is still unknown. A study from Muriel et al., reported that a sadB homolog in P. fluorescens binds c-di- GMP. The sadB homolog of P. aeruginosa was successfully purified and tested using SPR and thermal shift assay. The results showed no binding of c-di-GMP. Additionally, attempts were made to resolve the crystal structure of SadB. SadB crystals were obtained after screening commercial crystal screens and optimisation of the conditions but the resolution of the diffraction was not sufficient. Finally, SadB was found to localise in the cytoplasm using an anti-SadB antibody and Western blot and a SadB fluorescent fusion and super-resolution microscopy

Integration of vanHAX downstream of a ribosomal RNA operon restores vancomycin resistance in a susceptible Enterococcus faecium strain

During the genomic characterisation of Enterococcus faecium strains (n = 39) collected in a haematology ward, we identified an isolate (OI25), which contained vanA-type vancomycin resistance genes but was phenotypically susceptible to vancomycin. OI25 could revert to resistance when cultured in the presence of vancomycin and was thus considered to be vancomycin-variable. Long-read sequencing was used to identify structural variations within the vancomycin resistance region of OI25 and to uncover its resistance reversion mechanism. We found that OI25 has a reduced ability to positively regulate expression of the vanHAX genes in the presence of vancomycin, which was associated with the insertion of an IS6-family element within the promoter region and the first 50 bp of the vanR gene. The vancomycin-resistant revertant isolates constitutively expressed vanHAX genes at levels up to 36,000-fold greater than OI25 via co-transcription with a ribosomal RNA operon. The vancomycin-resistant revertants did not exhibit a significant growth defect. During VRE outbreaks, attention should be paid to contemporaneous vancomycin-susceptible strains as these may carry silent vancomycin resistance genes that can be activated through genomic rearrangements

Rapid expansion and international spread of M1 UK in the post-pandemic UK upsurge of Streptococcus pyogenes

The UK observed a marked increase in scarlet fever and invasive group A streptococcal infection in 2022 with severe outcomes in children and similar trends worldwide. Here we report lineage M1UK to be the dominant source of invasive infections in this upsurge. Compared with ancestral M1global strains, invasive M1UK strains exhibit reduced genomic diversity and fewer mutations in two-component regulator genes covRS. The emergence of M1UK is dated to 2008. Following a bottleneck coinciding with the COVID-19 pandemic, three emergent M1UK clades underwent rapid nationwide expansion, despite lack of detection in previous years. All M1UK isolates thus-far sequenced globally have a phylogenetic origin in the UK, with dispersal of the new clades in Europe. While waning immunity may promote streptococcal epidemics, the genetic features of M1UK point to a fitness advantage in pathogenicity, and a striking ability to persist through population bottlenecks

SadB is a Pseudomonas aeruginosa global regulator

Bacteria can adhere to almost any surface and form resistant biofilm communities. These pose a major clinical burden as they can form on medical devices such as catheters and are hard to prevent. Catheter- associated urinary tract infections (CAUTIs) are the most common nosocomial infection as they represent ~40% of all nosocomial infections. Hook et al., previously developed a series of polymers that prevent biofilm formation. Following a screen of 20,000 polymers and copolymers and subsequent scale-up, the hit acrylate polymer, EGdPEA has been clinically approved for the coating of urinary tract catheters and presents superior antibiofilm properties compared with silver-embedded silicone catheters. The bacterial biofilm resistance mechanism and surface sensing pathways involved are under investigation. In P. aeruginosa surface sensing includes the flagella, type IV pili and the wsp system. The present study sought to investigate SadB, an essential gene for biofilm formation and CheA, a histidine kinase of the chemotaxis system for their involvement in surface sensing. SadB is involved in the switch from reversible to irreversible surface attachment. Expression of sadB under a constitutive promoter overcomes the resistance of EGdPEA to biofilm formation. CheA is responsible for transmitting environmental signals to the cell and activating Pch phosphodiesterase, which is important in surface sensing. Biofilm and motility assays and monitoring on-surface gene expression were used as a test of potential involvement in surface sensing. In agreement with the literature, SadB was found to be essential for biofilm formation and to affect swarming motility. sadB was also found to be differentially expressed on the anti and pro-biofilm surfaces. In contrast, CheA affected swarming but did not affect biofilm formation on most polymer surfaces and glass. The promoter of cheA also controls the cheI operon with 13 more genes. The expression of the operon was found to be different on different surfaces but CheA is unlikely to be important in surface sensing. The literature and findings of the present study highlighted the importance of sadB in surface sensing, but the regulatory pathway was still unknown. Whole transcriptome analysis (RNAseq) was employed to investigate the downstream regulation of sadB in an untargeted manner. RT-PCR and phenotypic assays were used to validate the data. SadB was found to be a global regulator controlling up to 50% of the genome. sadB was shown to negatively affect the pqs system and subsequently the rhl system, rhamnolipid and pyocyanin production and the denitrification pathway. The levels of the PQS signal, pyocyanin and rhamnolipids were also shown to be affected by sadB. The denitrification pathway downregulation in the absence of sadB was shown to affect growth under microaerophilic conditions. Furthermore, sadB positively affected biofilm related targets such as Psl and c-di-GMP production. Advances were made in the present study in discovering the downstream targets of sadB that explain some of the observed phenotypes, however the function of SadB is still unknown. A study from Muriel et al., reported that a sadB homolog in P. fluorescens binds c-di- GMP. The sadB homolog of P. aeruginosa was successfully purified and tested using SPR and thermal shift assay. The results showed no binding of c-di-GMP. Additionally, attempts were made to resolve the crystal structure of SadB. SadB crystals were obtained after screening commercial crystal screens and optimisation of the conditions but the resolution of the diffraction was not sufficient. Finally, SadB was found to localise in the cytoplasm using an anti-SadB antibody and Western blot and a SadB fluorescent fusion and super-resolution microscopy

Antimicrobials and antimicrobial resistance (AAMR)

Antibiotics resistance is an emerging threat for the health systems worldwide.700000 deaths annually are attributed to antibiotic resistant pathogens. The predictions for 2050 are devastating. By 2050, 10 million deaths that are linked to antibiotic resistance are expected. The antibiotic resistance along with the shortage of new antibiotics have created an antibiotic crisis. One solution to antimicrobial resistance could be the investment in new innovative methods against antimicrobial resistance. In the present study, two research projects with innovative strategies were undertaken. The first project examined the route of transmission of Enterococcus faecium in a suspected outbreak using WGS. The second project concerned the characterisation of chemosensory proteins in the pathogen Pseudomonas aeruginosa in order to elucidate the mechanism by which the bacterium senses anti-attachment surface