Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital / Clostridioides difficile: caracterización de los toxinotipos circulantes en un hospital público de Argentina

Abstract Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

Resumen Clostridioides difficile es un anaerobio esporulado que se asocia con episodios de diarreas hospitalarias. Su virulencia se encuentra vinculada, principalmente, a las toxinas TcdA y TcdB, codificadas por sus respectivos genes, tcdA y tcdB, que son parte de un locus de patogenicidad (PaLoc). Nuestro objetivo fue caracterizar los toxinotipos de C. difficile circulantes en un hospital público. Los genes tcdA y tcdB fueron amplificados y digeridos con diferentes enzimas de restricción: EcoRI para tcdA; HincII y AccI para tcdB. Además, se evaluó la presencia de cdtB (gen de la toxina binaria B) y de las toxinas A y B (por dot blot), así como el efecto citotóxico de sobrenadantes de cultivo sobre células Vero. En conjunto, estos estudios revelaron tres toxinotipos circulantes según la clasificación de Rupnik: 0, I y VIII; el más prevalente fue el último. Aunque son necesarios más estudios (ej., secuenciación), es interesante notar que la presencia del toxinotipo I podría estar relacionada con la introducción de bacterias de diferente origen geográfico. En los pacientes infectados con el toxinotipo VIII, el análisis multivariante de los resultados de laboratorio mostró que los aislamientos asociados a decesos (GCD13, GCD14 y GCD22) estaban situados en regiones de los biplots relacionados con valores de laboratorio alterados al momento de la internación. En los otros pacientes, aunque no se observó correlación entre los valores de laboratorio al momento de la internación y la evolución clínica, los niveles de urea, creatinina y recuento de glóbulos blancos estuvieron correlacionados positivamente entre sí una vez diagnosticada la infección. Nuestro estudio revela la circulación de diferentes toxinotipos de C. difficile en un mismo hospital público. La variedad de toxinotipos puede originarse a partir de microorganismos preexistentes en la región, así como también por la introducción de bacterias provenientes de otras regiones geográficas. La existencia de microorganismos con diferente potencial patogénico es relevante para el control, el seguimiento y el tratamiento de las infecciones.

Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital.

Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

Conjugated Pt(II) Complexes as Luminescence-Switch-On Reporters Addressing the Microenvironment of Bacterial Biofilms.

In this work, the synthesis, structural and photophysical characterization of six phosphorescent H2O-soluble Pt(II) complexes are reported while addressing their emission maxima, photoluminescence quantum yields (ΦL), lifetimes (τ), aggregation tendency, and microenvironment sensitivity as a function of the substitution pattern on the main tridentate luminophore. Different ancillary ligands, namely, a trisulfonated phosphane and maltohexaose-conjugated pyridines (with or without amide bridges), were introduced and evaluated for the realization of switch-on-photoluminescent labels reporting on the microenvironment sensed in biofilms of Gram+ and Gram- models, namely, Staphylococcus aureus and Escherichia coli. With the aid of confocal luminescence micro(spectro)scopy, we observed that selected complexes specifically interact with the biofilms while leaving planktonic cells unlabeled. By using photoluminescence lifetime imaging microscopy, excited-state lifetimes within S. aureus biofilms were measured. The photoluminescence intensities were drastically boosted, and the excited state lifetimes were significantly prolonged upon binding to the viscous biofilm matrix, mainly due to the suppression of radiationless deactivation pathways upon shielding from physical quenching processes, such as interactions with solvent molecules and 3O2. The best performances were attained for non-aggregating complexes with maltohexaose targeting units and without amide bridges. Notably, in the absence of the maltodextrin, a hydrophobic adamantyl moiety suffices to attain a sizeable labeling capacity. Moreover, photoluminescence studies showed that selected complexes can also effectively interact with E. coli biofilms, where the bacterial cells are able to partially uptake the maltodextrin-based agents. In summary, the herein introduced concepts enable the development of specific biofilm reporters providing spatial resolution as well as lifetime- and spectrum-based readouts. Considering that most theragnostic agents reported so far mainly address metabolically active bacteria at the surface of biofilms but without reaching cells deeply immersed in the matrix, a new platform with a clear structure-property correlation is provided for the early detection of such bacterial arrays.

Probiotic Lactobacilli Isolated from Kefir Promote Down-Regulation of Inflammatory Lamina Propria T Cells from Patients with Active IBD.

Ulcerative colitis and Crohn's disease, the two main forms of inflammatory bowel disease (IBD), are immunologically mediated disorders. Several therapies are focused on activated T cells as key targets. Although Lactobacillus kefiri has shown anti-inflammatory effects in animal models, few studies were done using human mucosal T cells. The aim of this work was to investigate the immunomodulatory effects of this bacterium on intestinal T cells from patients with active IBD. Mucosal biopsies and surgical samples from IBD adult patients (n = 19) or healthy donors (HC; n = 5) were used. Lamina propria mononuclear cells were isolated by enzymatic tissue digestion, and entero-adhesive Escherichia coli-specific lamina propria T cells (LPTC) were expanded. The immunomodulatory properties of L. kefiri CIDCA 8348 strain were evaluated on biopsies and on anti-CD3/CD28-activated LPTC. Secreted cytokines were quantified by ELISA, and cell proliferation and viability were assessed by flow cytometry. We found that L. kefiri reduced spontaneous release of IL-6 and IL-8 from inflamed biopsies ex vivo. Activated LPTC from IBD patients showed low proliferative rates and reduced secretion of TNF-α, IL-6, IFN-γ and IL-13 in the presence of L. kefiri. In addition, L. kefiri induced an increased frequency of CD4+FOXP3+ LPTC along with high levels of IL-10. This is the first report showing an immunomodulatory effect of L. kefiri CIDCA 8348 on human intestinal cells from IBD patients. Understanding the mechanisms of interaction between probiotics and immune mucosal cells may open new avenues for treatment and prevention of IBD.

Comparative genomic analysis of Acinetobacter spp. plasmids originating from clinical settings and environmental habitats.

Bacteria belonging to the genus Acinetobacter have become of clinical importance over the last decade due to the development of a multi-resistant phenotype and their ability to survive under multiple environmental conditions. The development of these traits among Acinetobacter strains occurs frequently as a result of plasmid-mediated horizontal gene transfer. In this work, plasmids from nosocomial and environmental Acinetobacter spp. collections were separately sequenced and characterized. Assembly of the sequenced data resulted in 19 complete replicons in the nosocomial collection and 77 plasmid contigs in the environmental collection. Comparative genomic analysis showed that many of them had conserved backbones. Plasmid coding sequences corresponding to plasmid specific functions were bioinformatically and functionally analyzed. Replication initiation protein analysis revealed the predominance of the Rep_3 superfamily. The phylogenetic tree constructed from all Acinetobacter Rep_3 superfamily plasmids showed 16 intermingled clades originating from nosocomial and environmental habitats. Phylogenetic analysis of relaxase proteins revealed the presence of a new sub-clade named MOBQAci, composed exclusively of Acinetobacter relaxases. Functional analysis of proteins belonging to this group showed that they behaved differently when mobilized using helper plasmids belonging to different incompatibility groups.

Complete Genome Sequencing of Acinetobacter baumannii Strain K50 Discloses the Large Conjugative Plasmid pK50a Encoding Carbapenemase OXA-23 and Extended-Spectrum ß-Lactamase GES-11.

Multidrug-resistant (MDR) Acinetobacter baumannii strains appeared as serious emerging nosocomial pathogens in clinical environments and especially in intensive care units (ICUs). A. baumannii strain K50, recovered from a hospitalized patient in Kuwait, exhibited resistance to carbapenems and additionally to ciprofloxacin, chloramphenicol, sulfonamides, amikacin, and gentamicin. Genome sequencing revealed that the strain possesses two plasmids, pK50a (79.6 kb) and pK50b (9.5 kb), and a 3.75-Mb chromosome. A. baumannii K50 exhibits an average nucleotide identity (ANI) of 99.98% to the previously reported Iraqi clinical isolate AA-014, even though the latter strain lacked plasmid pK50a. Strain K50 belongs to sequence type 158 (ST158) (Pasteur scheme) and ST499 (Oxford scheme). Plasmid pK50a is a member of the Aci6 (replication group 6 [RG6]) group of Acinetobacter plasmids and carries a conjugative transfer module and two antibiotic resistance gene regions. The transposon Tn2008 carries the carbapenemase gene blaOXA-23, whereas a class 1 integron harbors the resistance genes blaGES-11, aacA4, dfrA7, qacEΔ1, and sul1, conferring resistance to all ß-lactams and reduced susceptibility to carbapenems and resistance to aminoglycosides, trimethoprim, quaternary ammonium compounds, and sulfamethoxazole, respectively. The class 1 integron is flanked by MITEs (miniature inverted-repeat transposable elements) delimiting the element at its insertion site.

Novel tnpR-based transposable promoter traps suitable for RIVET studies in different gram-negative bacteria.

The preparation of plasmid-borne RIVET libraries can be troublesome when high genomic coverages are needed. We present here the construction and functional validation of a new set of miniTn5 promoter traps to generate tnpR-based RIVET libraries. The ability to generate tnpR transcriptional fusions by transposition will significantly facilitate the setup of RIVET studies in those bacteria where Tn5 transposition is operative.

First genomic analysis of the broad-host-range Rhizobium sp. LPU83 strain, a member of the low-genetic diversity Oregon-like Rhizobium sp. group.

Alfalfa (Medicago sativa) is the most cultivated forage legume for cattle and animal feeding, occupying about 32 million hectares over the world. Management of the N2-fixing symbiosis of this plant to maximize crop production is therefore an important objective. A fundamental constraint to this aim emerges when a moderately low soil pH hampers the establishment of an effective symbiosis with indigenous and/or inoculated rhizobia. Besides the association of alfalfa with Ensifer (Sinorhizobium) meliloti, this legume is able to establish a symbiosis with Ensifer (Sinorhizobium) medicae and with less characterized types of rhizobia, such as the Oregon-like strains, Rhizobium sp. Or191 initially isolated in the USA, and the Rhizobium sp. LPU83 strain, from Argentina. These strains are acid-tolerant, highly competitive for acidic-soil-alfalfa nodulation, but inefficient for biological nitrogen fixation with alfalfa. These features position the Oregon-like rhizobia as strains of potential risk in agricultural soils compared with the efficient symbiont E. meliloti. Moreover, the collected genetic information has revealed that the genomic structure of these rhizobial isolates is complex in terms of sequence similarities shared with other rhizobia. Such a "patched" genetic composition has obviously imposed severe restrictions to the classical taxonomy of these rhizobia. In this work we summarize the accumulated knowledge about the Oregon-like rhizobia and present a phylogenetic analysis based on genome sequence data of Rhizobium sp. LPU83 obtained by a high-throughput sequencing on the Genome Sequencer FLX Titanium platform. The accessibility of the complete genomic sequence will release up more experimental possibilities since this information will then enable biochemical studies as well as proteomics and transcriptomics approaches.