Comparative Genomic Analysis of Enterococci across Sectors of the One Health Continuum.

Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3')-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade 'A' and 'B', with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.

Resistance determinants and their genetic context in enterobacteria from a longitudinal study of pigs reared under various husbandry conditions.

Pigs are major reservoirs of resistant Enterobacteriaceae that can reach humans through consumption of contaminated meat or vegetables grown in manure-fertilized soil. Samples were collected from sows during lactation and their piglets at five time points spanning the production cycle. Cefotaxime-resistant bacteria were quantified and isolated from feed, feces, manures and carcasses of pigs reared with penicillin-using or antibiotic-free husbandries. The isolates were characterized by antibiotic susceptibility testing, whole genome sequencing and conjugation assays. The extended spectrum ß-lactamase (ESBL) phenotype was more frequent in isolates originating from antibiotic-free animals, while the bacteria isolated from penicillin-using animals were on average resistant to a greater number of antibiotics. The ESBL-encoding genes identified were bla CTX-M-1, bla CTX-M-15 and bla CMY-2 and they co-localised on plasmids with various genes encoding resistance to ß-lactams, co-trimoxazole, phenicols and tetracycline, all antibiotics used in pig production. Groups of genes conferring the observed resistance and the mobile elements disseminating multidrug resistance were determined. The observed resistance to ß-lactams was mainly due to the complementary actions of penicillin-binding proteins, an efflux pump and ß-lactamases. Most resistance determinants were shared by animals raised with or without antimicrobials. This suggests a key contribution of indigenous enterobacteria maternally transmitted along the sow lineage, regardless of antimicrobial use. It is unclear if the antimicrobial resistance observed in the enterobacteria populations of the commercial pig herds studied were present before the use of antibiotics, or the extent to which historical antimicrobial use exerted a selective pressure defining the resistant bacterial populations in farms using penicillin prophylaxis.Importance: Antimicrobial resistance is a global threat that needs to be fought on numerous fronts along the One Health continuum. Vast quantities of antimicrobials are used in agriculture to ensure animal welfare and productivity, and are arguably a driving force for the persistence of environmental and food-borne resistant bacteria. This study evaluated the impact of conventional, organic and other antibiotic-free husbandry practices on the frequency and nature of antimicrobial resistance genes and multidrug resistant enterobacteria. It provides knowledge about the relative contribution of specific resistance determinants to observed antibiotic resistance. It also showed the clear co-selection of genes coding for extended-spectrum beta-lactamases and genes coding for the resistance to antibiotics commonly used for prophylaxis or in curative treatments in pig operations.

A λ Cro-Like Repressor Is Essential for the Induction of Conjugative Transfer of SXT/R391 Elements in Response to DNA Damage.

UNLABELLED: Integrative and conjugative elements (ICEs) of the SXT/R391 family are the main contributors to acquired multidrug resistance in the seventh pandemic lineage of Vibrio cholerae, the etiological agent of the diarrheal disease cholera. Conjugative transfer of SXT/R391 ICEs is triggered by antibiotics and agents promoting DNA damage through RecA-dependent autoproteolysis of SetR, an ICE-encoded λ CI-like repressor. Here, we describe the role of CroS, a distant λ Cro homolog, as a key component contributing to the regulation of expression of the activator SetCD that orchestrates the expression of the conjugative transfer genes. We show that deletion of croS abolishes the SOS response-dependent induction of SXT despite the presence of a functional setR gene. Using quantitative reverse transcription-PCR and lacZ reporter assays, we also show that CroS represses setR and setCD expression by binding to operator sites shared with SetR. Furthermore, we provide evidence of an additional operator site bound by SetR and CroS. Finally, we show that SetCD expression generates a positive feedback loop due to SXT excision and replication in a fraction of the cell population. Together, these results refine our understanding of the genetic regulation governing the propagation of major vectors of multidrug resistance. IMPORTANCE: Healthcare systems worldwide are challenged by an alarming drug resistance crisis caused by the massive and rapid propagation of antibiotic resistance genes and the associated emergence of multidrug-resistant pathogenic bacteria. SXT/R391 ICEs contribute to this phenomenon not only in clinical and environmental vibrios but also in several members of the family Enterobacteriaceae. We have identified and characterized here the regulator CroS as a key factor in the stimulation of conjugative transfer of these ICEs in response to DNA-damaging agents. We have also untangled conflicting evidence regarding autoactivation of transfer by the master activator of SXT/R391 ICEs, SetCD. Discovery of CroS provides a clearer and more complete understanding of the regulatory network that governs the dissemination of SXT/R391 ICEs in bacterial populations.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes.

Electrophoretic mobility shift assays (EMSA) have proven their usefulness for studying interactions between biological molecules. In the present protocol, a purified protein of interest is mixed with a 5'-end radiolabeled DNA probe. The bound complexes are separated by electrophoretic migration through a polyacrylamide gel and detected with a phosphorimager. The applications of EMSA are diverse, from thermodynamic and kinetic analyses to observation of bending and other conformational changes, stoichiometric inferences, or insights into cooperative protein binding.

A diaminopimelic acid auxotrophic Escherichia coli donor provides improved counterselection following intergeneric conjugation with actinomycetes.

Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.

Transfer activation of SXT/R391 integrative and conjugative elements: unraveling the SetCD regulon.

Integrative and conjugative elements (ICEs) of the SXT/R391 family have been recognized as key drivers of antibiotic resistance dissemination in the seventh-pandemic lineage of Vibrio cholerae. SXT/R391 ICEs propagate by conjugation and integrate site-specifically into the chromosome of a wide range of environmental and clinical Gammaproteobacteria. SXT/R391 ICEs bear setC and setD, two conserved genes coding for a transcriptional activator complex that is essential for activation of conjugative transfer. We used chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) to characterize the SetCD regulon of three representative members of the SXT/R391 family. We also identified the DNA sequences bound by SetCD in MGIVflInd1, a mobilizable genomic island phylogenetically unrelated to SXT/R391 ICEs that hijacks the conjugative machinery of these ICEs to drive its own transfer. SetCD was found to bind a 19-bp sequence that is consistently located near the promoter -35 element of SetCD-activated genes, a position typical of class II transcriptional activators. Furthermore, we refined our understanding of the regulation of excision from and integration into the chromosome for SXT/R391 ICEs and demonstrated that de novo expression of SetCD is crucial to allow integration of the incoming ICE DNA into a naive host following conjugative transfer.

DNA-damaging agents induce the RecA-independent homologous recombination functions of integrating conjugative elements of the SXT/R391 family.

Integrating conjugative elements (ICEs) of the SXT/R391 family are major contributors to the spread of antibiotic resistance genes. These elements also catalyze their own diversity by promoting inter-ICE recombination through the action of the RecA-independent homologous recombination system that they encode. Here, we report that expression of this recombination system, which consists of the single-stranded DNA annealing protein Bet and the exonuclease Exo, is induced by DNA-damaging agents via ICE-encoded transcriptional regulators. We show that the bet and exo genes are part of a large polycistronic transcript that contains many conserved ICE genes that are not involved in the main integration/excision and conjugative transfer processes. We show that although the recombination genes are highly transcribed, their translation is subject to additional strong regulatory mechanisms. We also show that an ICE-encoded putative single-stranded DNA binding protein (Ssb) limits hybrid ICE formation. Finally, a thorough in silico analysis reveals that orthologues of Bet and Exo are widely distributed in bacterial strains belonging to very distantly related bacterial species and are carried by various mobile genetic elements. Phylogenetic analyses suggest that the annealing proteins and exonucleases that compose these systems sometimes have different evolutionary origins, underscoring the strong selective pressure to maintain the functionality of these unrelated cooperating proteins.

Dynamics of the SetCD-regulated integration and excision of genomic islands mobilized by integrating conjugative elements of the SXT/R391 family.

Mobilizable genomic islands (MGIs) are small genomic islands that are mobilizable by SXT/R391 integrating conjugative elements (ICEs) due to similar origins of transfer. Their site-specific integration and excision are catalyzed by the integrase that they encode, but their conjugative transfer entirely depends upon the conjugative machinery of SXT/R391 ICEs. In this study, we report the mechanisms that control the excision and integration processes of MGIs. We found that while the MGI-encoded integrase Int(MGI) is sufficient to promote MGI integration, efficient excision from the host chromosome requires the combined action of Int(MGI) and of a novel recombination directionality factor, RdfM. We determined that the genes encoding these proteins are activated by SetCD, the main transcriptional activators of SXT/R391 ICEs. Although they share the same regulators, we found that unlike rdfM, int(MGI) has a basal level of expression in the absence of SetCD. These findings explain how an MGI can integrate into the chromosome of a new host in the absence of a coresident ICE and shed new light on the cross talk that can occur between mobilizable and mobilizing elements that mobilize them, helping us to understand part of the rules that dictate horizontal transfer mechanisms.

Properties of CsnR, the transcriptional repressor of the chitosanase gene, csnA, of Streptomyces lividans.

A palindromic sequence is present in the intergenic region preceding the chitosanase gene csnA (SSPG_06922) of Streptomyces lividans TK24. This sequence was also found in front of putative chitosanase genes in several other actinomycete genomes and upstream genes encoding putative transcriptional regulators of the ROK family, including csnR (SSPG_04872) in S. lividans. The latter was examined as a possible transcriptional regulator (CsnR) of chitosanase gene expression. In vitro, purified CsnR bound strongly to the palindromic sequences of the csnA and csnR genes (equilibrium dissociation constant [K(D)] = 0.032 and 0.040 nM, respectively). Binding was impaired in the presence of chitosan oligosaccharides and d-glucosamine, and chitosan dimer was found to be the best effector, as determined by an equilibrium competition experiment and 50% inhibitory concentration (IC(50)) determination, while glucose, N-acetyl-glucosamine, and galactosamine had no effect. In vivo, comparison of the S. lividans wild type and ΔCsnR strains using ß-lactamase reporter genes showed that CsnR represses the expression of csnA and of its own gene, which was confirmed by quantitative PCR (qPCR). CsnR is localized at the beginning of a gene cluster, possibly an operon, the organization of which is conserved through many actinomycete genomes. The CsnR-mediated chitosanase regulation mechanism seems to be widespread among actinomycetes.

Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan.

Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a chitosanase-overproducing strain. This is the first genetic evidence for the involvement of chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.