ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

MogR Is a Ubiquitous Transcriptional Repressor Affecting Motility, Biofilm Formation and Virulence in Bacillus thuringiensis.

Flagellar motility is considered an important virulence factor in different pathogenic bacteria. In Listeria monocytogenes the transcriptional repressor MogR regulates motility in a temperature-dependent manner, directly repressing flagellar- and chemotaxis genes. The only other bacteria known to carry a mogR homolog are members of the Bacillus cereus group, which includes motile species such as B. cereus and Bacillus thuringiensis as well as the non-motile species Bacillus anthracis, Bacillus mycoides and Bacillus pseudomycoides. Furthermore, the main motility locus in B. cereus group bacteria, carrying the genes for flagellar synthesis, appears to be more closely related to L. monocytogenes than to Bacillus subtilis, which belongs to a separate phylogenetic group of Bacilli and does not carry a mogR ortholog. Here, we show that in B. thuringiensis, MogR overexpression results in non-motile cells devoid of flagella. Global gene expression profiling showed that 110 genes were differentially regulated by MogR overexpression, including flagellar motility genes, but also genes associated with virulence, stress response and biofilm lifestyle. Accordingly, phenotypic assays showed that MogR also affects cytotoxicity and biofilm formation in B. thuringiensis. Overexpression of a MogR variant mutated in two amino acids within the putative DNA binding domain restored phenotypes to those of an empty vector control. In accordance, introduction of these mutations resulted in complete loss in MogR binding to its candidate flagellar locus target site in vitro. In contrast to L. monocytogenes, MogR appears to be regulated in a growth-phase dependent and temperature-independent manner in B. thuringiensis 407. Interestingly, mogR was found to be conserved also in non-motile B. cereus group species such as B. mycoides and B. pseudomycoides, which both carry major gene deletions in the flagellar motility locus and where in B. pseudomycoides mogR is the only gene retained. Furthermore, mogR is expressed in non-motile B. anthracis. Altogether this provides indications of an expanded set of functions for MogR in B. cereus group species, beyond motility regulation. In conclusion, MogR constitutes a novel B. thuringiensis pleiotropic transcriptional regulator, acting as a repressor of motility genes, and affecting the expression of a variety of additional genes involved in biofilm formation and virulence.

Bacillus cereus biofilm formation on central venous catheters of hospitalised cardiac patients.

Formation of bacterial biofilms is a risk with many in situ medical devices. Biofilm-forming Bacillus species are associated with potentially life-threatening catheter-related blood stream infections in immunocompromised patients. Here, bacteria were isolated from biofilm-like structures within the lumen of central venous catheters (CVCs) from two patients admitted to cardiac hospital wards. Isolates belonged to the Bacillus cereus group, exhibited strong biofilm formation propensity, and mapped phylogenetically close to the B. cereus emetic cluster. Together, whole genome sequencing and quantitative PCR confirmed that the isolates constituted the same strain and possessed a range of genes important for and up-regulated during biofilm formation. Antimicrobial susceptibility testing demonstrated resistance to trimethoprim-sulphamethoxazole, clindamycin, penicillin and ampicillin. Inspection of the genome revealed several chromosomal ß-lactamase genes and a sulphonamide resistant variant of folP. This study clearly shows that B. cereus persisting in hospital ward environments may constitute a risk factor from repeated contamination of CVCs.

Bacillus thuringiensis CbpA is a collagen binding cell surface protein under c-di-GMP control.

Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in Bacillus cereus group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in B. thuringiensis. C-di-GMP can exert its function as a second messenger via riboswitch binding, and a functional c-di-GMP-responsive riboswitch has been found upstream of cbpA in various B. cereus group strains. Protein signature recognition predicted CbpA to be a cell wall-anchored surface protein with a fibrinogen or collagen binding domain. The aim of this study was to identify the binding ligand of CbpA and the function of CbpA in cellular processes that are part of the B. cereus group c-di-GMP regulatory network. By global gene expression profiling cbpA was found to be down-regulated in a cdgF deletion mutant, and cbpA exhibited maximum expression in early exponential growth. Contrary to the wild type, a ΔcbpA deletion mutant showed no binding to collagen in a cell adhesion assay, while a CbpA overexpression strain exhibited slightly increased collagen binding compared to the control. For both fibrinogen and fibronectin there was however no change in binding activity compared to controls, and CbpA did not appear to contribute to binding to abiotic surfaces (polystyrene, glass, steel). Also, the CbpA overexpression strain appeared to be less motile and showed a decrease in biofilm formation compared to the control. This study provides the first experimental proof that the binding ligand of the c-di-GMP regulated adhesin CbpA is collagen.

The putative drug efflux systems of the Bacillus cereus group.

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70-80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current knowledge of the small molecule efflux pumps encoded by the B. cereus group and suggest the likely functions of numerous uncharacterised pumps.