Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Gut Microbiota and Colonization Resistance against Bacterial Enteric Infection.

The gut microbiome is critical in providing resistance against colonization by exogenous microorganisms. The mechanisms via which the gut microbiota provide colonization resistance (CR) have not been fully elucidated, but they include secretion of antimicrobial products, nutrient competition, support of gut barrier integrity, and bacteriophage deployment. However, bacterial enteric infections are an important cause of disease globally, indicating that microbiota-mediated CR can be disturbed and become ineffective. Changes in microbiota composition, and potential subsequent disruption of CR, can be caused by various drugs, such as antibiotics, proton pump inhibitors, antidiabetics, and antipsychotics, thereby providing opportunities for exogenous pathogens to colonize the gut and ultimately cause infection. In addition, the most prevalent bacterial enteropathogens, including Clostridioides difficile, Salmonella enterica serovar Typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, Campylobacter jejuni, Vibrio cholerae, Yersinia enterocolitica, and Listeria monocytogenes, can employ a wide array of mechanisms to overcome colonization resistance. This review aims to summarize current knowledge on how the gut microbiota can mediate colonization resistance against bacterial enteric infection and on how bacterial enteropathogens can overcome this resistance.

Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis.

In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.

Genome-based insights into the evolution of enterococci.

It is now 15 years since the first genome of a free-living organism was sequenced. Subsequent to this milestone, a veritable avalanche of genome sequence data has revolutionized many aspects of microbiology. In this review, we discuss recent progress on the genomics of Enterococcus faecalis and Enterococcus faecium, which are the two enterococcal species that cause the large majority of enterococcal infections. We focus on the genome-based analysis of enterococcal diversity and phylogeny. Studies based on comparative genome hybridization have shown that both species exhibit considerable inter-strain genomic diversity, which is mainly linked to the variable presence of phages, plasmids, pathogenicity islands and conjugative elements. We also discuss how the advent of next-generation sequencing technologies allows for a comprehensive characterization of the gene repertoire of multiple isolates, which can be used for extremely robust analyses of diversity and population structure.

Acid-adapted Listeria monocytogenes displays enhanced tolerance against the lantibiotics nisin and lacticin 3147.

Log-phase Listeria monocytogenes cells become tolerant to a variety of environmental stresses following acid adaptation at pH 5.5. We demonstrated that adapted cells also exhibit increased tolerance to nisin and, to a lesser extent, lacticin 3147. At nisin concentrations of 100 and 200 IU/ml the survival of acid-adapted cells was approximately 10-fold greater than nonadapted cells. However, acid adaptation had only a moderate effect on the tolerance of L. monocytogenes to lacticin 3147, a phenomenon that possibly reflects the distinct mode of action of this bacteriocin. Analysis of the fatty acid composition of the bacterial membrane indicated that straight-chain fatty acids C14:0 and C16:0 were significantly increased in acid-adapted cells while levels of C18:0 decreased. The results indicate that stress mechanisms that are induced in mildly acidic conditions provide protection against the antimicrobial action of bacteriocins. This increased resistance of acid-adapted L. monocytogenes could cause increased survival of this pathogen in food products in which nisin or other bacteriocins are used as preservatives.

The use of hydrolytic enzymes as adjuvant therapy in AIDS/ARC/LAS patients.

The purpose of this paper is to discuss a hypothesis corroborating the use of hydrolytic enzymes in AIDS/ARC/LAS patients, showing high levels of circulating immune complexes (CIC). Other diseases, revealing high CIC-levels as well, may be improved or even cured by eliminating CIC. The results obtained from experiences of CIC-elimination by hydrolytic enzymes are presented below. As may be determined from the data available, hydrolytic enzymes should be included in the therapy of HIV-positive patients. Clinical trials are being continued.