Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens.

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using in vitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy.

Detecting envelope stress by monitoring ß-barrel assembly.

The cell envelope protects bacteria from their surroundings. Defects in its integrity or assembly are sensed by signal transduction systems, allowing cells to rapidly adjust. The Rcs phosphorelay responds to outer membrane (OM)- and peptidoglycan-related stress in enterobacteria. We elucidated how the OM lipoprotein RcsF, the upstream Rcs component, senses envelope stress and activates the signaling cascade. RcsF interacts with BamA, the major component of the ß-barrel assembly machinery. In growing cells, BamA continuously funnels RcsF through the ß-barrel OmpA, displaying RcsF on the cell surface. This process spatially separates RcsF from the downstream Rcs component, which we show is the inner membrane protein IgaA. The Rcs system is activated when BamA fails to bind RcsF and funnel it to OmpA. Newly synthesized RcsF then remains periplasmic, interacting with IgaA to activate the cascade. Thus RcsF senses envelope damage by monitoring the activity of the Bam machinery.

Bactericidal effect of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against carbapenem-resistant Acinetobacter baumannii.

In this study, we evaluated the antimicrobial activity of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against seven Acinetobacter baumannii strains, the majority of which were clinically relevant carbapenem-resistant A. baumannii strains. We observed the inhibitory activity of 18 (out of 114) sponge-isolated bacterial strains against all A. baumanii strains using medium-throughput solid agar overlay assays. These inhibitory strains belonged to the genera Lactococcus, Pseudomonas, and Vagococcus. In addition, this antimicrobial activity was validated through a liquid co-cultivation challenge using an inhibitory strain of each genus and a green fluorescent protein-tagged A. baumanii strain. Fluorescence measurements indicated that the growth of A. baumanii was inhibited by the sponge isolates. In addition, the inability of A. baumanii to grow after spreading the co-cultures on solid medium allowed us to characterize the activity of the sponge isolates as bactericidal. In conclusion, this study demonstrates that marine sponges are a reservoir of bacteria that deserves to be tapped for antibiotic discovery against A. baumanii.

Genomic Analysis of a Strain Collection Containing Multidrug-, Extensively Drug-, Pandrug-, and Carbapenem-Resistant Modern Clinical Isolates of Acinetobacter baumannii.

In this study, we characterize a new collection that comprises multidrug-resistant (MDR), extensively drug-resistant (XDR), pandrug-resistant (PDR), and carbapenem-resistant modern clinical isolates of Acinetobacter baumannii collected from hospitals through national microbiological surveillance in Belgium. Bacterial isolates (n = 43) were subjected to whole-genome sequencing (WGS), combining Illumina (MiSeq) and Nanopore (MinION) technologies, from which high-quality genomes (chromosome and plasmids) were de novo assembled. Antimicrobial susceptibility testing was performed along with genome analyses, which identified intrinsic and acquired resistance determinants along with their genetic environments and vehicles. Furthermore, the bacterial isolates were compared to the most prevalent A. baumannii sequence type 2 (ST2) (Pasteur scheme) genomes available from the BIGSdb database. Of the 43 strains, 40 carried determinants of resistance to carbapenems; blaOXA-23 (n = 29) was the most abundant acquired antimicrobial resistance gene, with 39 isolates encoding at least two different types of OXA enzymes. According to the Pasteur scheme, the majority of the isolates were globally disseminated clones of ST2 (n = 25), while less frequent sequence types included ST636 (n = 6), ST1 (n = 4), ST85 and ST78 (n = 2 each), and ST604, ST215, ST158, and ST10 (n = 1 each). Using the Oxford typing scheme, we identified 22 STs, including two novel types (ST2454 and ST2455). While the majority (26/29) of blaOXA-23 genes were chromosomally carried, all blaOXA-72 genes were plasmid borne. Our results show the presence of high-risk clones of A. baumannii within Belgian health care facilities with frequent occurrences of genes encoding carbapenemases, highlighting the crucial need for constant surveillance.

Polar growth in the Alphaproteobacterial order Rhizobiales.

Elongation of many rod-shaped bacteria occurs by peptidoglycan synthesis at discrete foci along the sidewall of the cells. However, within the Rhizobiales, there are many budding bacteria, in which new cell growth is constrained to a specific region. The phylogeny of the Rhizobiales indicates that this mode of zonal growth may be ancestral. We demonstrate that the rod-shaped bacterium Agrobacterium tumefaciens grows unidirectionally from the new pole generated after cell division and has an atypical peptidoglycan composition. Polar growth occurs under all conditions tested, including when cells are attached to a plant root and under conditions that induce virulence. Finally, we show that polar growth also occurs in the closely related bacteria Sinorhizobium meliloti, Brucella abortus, and Ochrobactrum anthropi. We find that unipolar growth is an ancestral and conserved trait among the Rhizobiales, which includes important mutualists and pathogens of plants and animals.

Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.

BtaE, an adhesin that belongs to the trimeric autotransporter family, is required for full virulence and defines a specific adhesive pole of Brucella suis.

Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.

The intracellular life of Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic bacterium responsible for nosocomial and community-acquired infections. This pathogen is globally disseminated and associated with high levels of antibiotic resistance, which makes it an important threat to human health. Recently, new evidence showed that several A. baumannii isolates can survive and proliferate within eukaryotic professional and/or nonprofessional phagocytic cells, with in vivo consequences. This review provides updated information and describes the tools that A. baumannii possesses to adhere, colonize, and replicate in host cells. Additionally, we emphasize the high genetic and phenotypic heterogeneity detected amongst A. baumannii isolates and its impact on the bacterial intracellular features. We also discuss the need for standardized methods to characterize this pathogen robustly and consequently consider some strains as facultative intracellular bacteria.

Phenotypic Characterization and Heterogeneity among Modern Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogenic bacterium prioritized by WHO and CDC because of its increasing antibiotic resistance. Heterogeneity among strains represents the hallmark of A. baumannii bacteria. We wondered to what extent extensively used strains, so-called reference strains, reflect the dynamic nature and intrinsic heterogeneity of these bacteria. We analyzed multiple phenotypic traits of 43 nonredundant, modern, and multidrug-resistant, extensively drug-resistant, and pandrug-resistant clinical isolates and broadly used strains of A. baumannii. Comparison of these isolates at the genetic and phenotypic levels confirmed a high degree of heterogeneity. Importantly, we observed that a significant portion of modern clinical isolates strongly differs from several historically established strains in the light of colony morphology, cellular density, capsule production, natural transformability, and in vivo virulence. The significant differences between modern clinical isolates of A. baumannii and established strains could hamper the study of A. baumannii, especially concerning its virulence and resistance mechanisms. Hence, we propose a variable collection of modern clinical isolates that are characterized at the genetic and phenotypic levels, covering a wide range of the phenotypic spectrum, with six different macrocolony type groups, from avirulent to hypervirulent phenotypes, and with naturally noncapsulated to hypermucoid strains, with intermediate phenotypes as well. Strain-specific mechanistic observations remain interesting per se, and established "reference" strains have undoubtedly been shown to be very useful to study basic mechanisms of A. baumannii biology. However, any study based on a specific strain of A. baumannii should be compared to modern and clinically relevant isolates. IMPORTANCE Acinetobacter baumannii is a bacterium prioritized by the CDC and WHO because of its increasing antibiotic resistance, leading to treatment failures. The hallmark of this pathogen is the high heterogeneity observed among isolates, due to a very dynamic genome. In this context, we tested if a subset of broadly used isolates, considered "reference" strains, was reflecting the genetic and phenotypic diversity found among currently circulating clinical isolates. We observed that the so-called reference strains do not cover the whole diversity of the modern clinical isolates. While formerly established strains successfully generated a strong base of knowledge in the A. baumannii field and beyond, our study shows that a rational choice of strain, related to a specific biological question, should be taken into consideration. Any data obtained with historically established strains should also be compared to modern and clinically relevant isolates, especially concerning drug screening, resistance, and virulence contexts.

The histidine kinase PdhS controls cell cycle progression of the pathogenic alphaproteobacterium Brucella abortus.

Bacterial differentiation is often associated with the asymmetric localization of regulatory proteins, such as histidine kinases. PdhS is an essential and polarly localized histidine kinase in the pathogenic alphaproteobacterium Brucella abortus. After cell division, PdhS is asymmetrically segregated between the two sibling cells, highlighting a differentiation event. However, the function(s) of PdhS in the B. abortus cell cycle remains unknown. We used an original approach, the pentapeptide scanning mutagenesis method, to generate a thermosensitive allele of pdhS. We report that a B. abortus strain carrying this pdhS allele displays growth arrest and an altered DivK-yellow fluorescent protein (YFP) polar localization at the restrictive temperature. Moreover, the production of a nonphosphorylatable PdhS protein or truncated PdhS proteins leads to dominant-negative effects by generating morphological defects consistent with the inhibition of cell division. In addition, we have used a domain mapping approach combined with yeast two-hybrid and fluorescence microscopy methods to better characterize the unusual PdhS sensory domain. We have identified a fragment of the PdhS sensory domain required for protein-protein interaction (amino acids [aa] 210 to 434), a fragment sufficient for polar localization (aa 1 to 434), and a fragment (aa 527 to 661) whose production in B. abortus correlates with the generation of cell shape alterations. The data support a model in which PdhS acts as an essential regulator of cell cycle progression in B. abortus and contribute to a better understanding of the differentiation program inherited by the two sibling cells.