Draft genome of the Klebsiella pneumoniae 24Kpn33 and complete sequence of its pCOL-1, a plasmid related to the blaKPC-2 acquisition in Pseudomonas aeruginosa.

We report the draft genome of a clinical multi-resistant Klebsiella pneumoniae (24Kpn33) isolate, whose genome (5.7 Mbp) harbored 17 antibiotic resistance genes, including blaKPC-2. Notably, this gene was mobilized within the IncP-6 pCOL-1 plasmid, the first genetic platform related to the acquisition and dissemination of the blaKPC-2 in Pseudomonas aeruginosa.

Association of Root Biofilm Bacteriome with Root Caries Lesion Severity and Activity.

INTRODUCTION: This research aimed to assess the association of root biofilm bacteriome with root caries lesion severity and activity in institutionalised Colombian elderlies and was conducted to gather data on the root caries bacteriome in this population. METHODS: A bacteriome evaluation of biofilm samples from sound and carious root surfaces was performed. Root caries was categorised (ICDAS Root criteria) based on severity (sound surfaces, initial: non-cavitated, moderate/extensive combined: cavitated) and activity status (active and inactive). DNA was extracted and the V4 region of the 16S rRNA gene was sequenced; afterwards the classification of features was conducted employing amplicon sequence variants and taxonomic assignment via the Human Oral Microbiome Database (HOMD). Bacterial richness, diversity (Simpson's and Shannon's indices), and relative abundance estimation were assessed and compared based on root caries severity and activity status (including Sound surfaces). RESULTS: A total of 130 biofilm samples were examined: sound (n = 45) and with root caries lesions (n = 85; by severity: initial: n = 41; moderate/extensive: n = 44; by activity: active: n = 60; inactive: n = 25). Species richness was significantly lower in biofilms from moderate/extensive and active groups compared to sound sites. There was a higher relative abundance of species like Lechtotricia wadei, Capnocytophaga granulosa, Cardiobacterium valvarum, Porphyromonas pasteri - in sound sites; Dialister invisus, Streptococcus mutans, Pseudoramibacter alactolyticus and Bacteroidetes (G-5) bacterium 511 - in moderate/extensive lesions, and Fusobacterium nucleatum subsp. animalis, Prevotella denticola, Lactobacillus fermentum, Saccharibacteria (TM7) (G-5)bacterium HMT 356 - in active lesions. CONCLUSION: Root caries bacteriome exhibited differences in species proportions between the compared groups. Specifically, cavitated caries lesions and active caries lesions showed higher relative abundance of acidogenic bacteria.

Differential gut microbiome in spondyloarthritis patients associated to Blastocystis colonization.

The role of Blastocystis in intestinal health is an open controversy, and little is known about the potential effect of this microorganism in autoinflammatory diseases such as spondyloarthritis (SpA). Here, we analyzed the gut microbiome of 36 SpA patients and 13 control individuals and demonstrated that the richness, diversity, and taxonomic composition between these two groups are different. We also showed that colonization by Blastocystis in control individuals increases the richness and diversity of the intestinal microbiome, whereas in SpA patients, it does not seem to have any impact. This may reflect a potential role of Blastocystis in sculpting the gut microbiome architecture in control individuals, whereas in subjects with SpA, the modulation of the microbiome may be governed by disease-dependent factors that cannot be overcome by Blastocystis. Regarding taxonomic characterization, SpA patients colonized by Blastocystis showed significant increases in the phylum Pseudomonadota, class Gammaproteobacteria, family Succinivibrionaceae, and genus Succinivibrio. Simultaneously, there were significant increases in the class Bacilli, order Lactobacillales, families Lactobacillaceae and Clostridiaceae, and genera Lactobacillus and Clostridium in non-colonized SpA patients. On the other hand, PICRUSt analysis in Blastocystis-positive SpA patients showed elevations in pathways that may enhance antioxidant capacities and alleviate intestinal inflammation, while Blastocystis-negative SpA patients showed significant changes in pathways that promote cell division/proliferation and can lead to larger changes in the gut microbiome. Our analyses lead us to believe that these changes in the gut microbiome of SpA patients may trigger protective mechanisms as an initial response to inflammation in an attempt to restore balance in the intestinal environment.

Worldwide Dissemination of blaKPC Gene by Novel Mobilization Platforms in Pseudomonas aeruginosa: A Systematic Review.

The dissemination of blaKPC-harboring Pseudomonas aeruginosa (KPC-Pa) is considered a serious public health problem. This study provides an overview of the epidemiology of these isolates to try to elucidate novel mobilization platforms that could contribute to their worldwide spread. A systematic review in PubMed and EMBASE was performed to find articles published up to June 2022. In addition, a search algorithm using NCBI databases was developed to identify sequences that contain possible mobilization platforms. After that, the sequences were filtered and pair-aligned to describe the blaKPC genetic environment. We found 691 KPC-Pa isolates belonging to 41 different sequence types and recovered from 14 countries. Although the blaKPC gene is still mobilized by the transposon Tn4401, the non-Tn4401 elements (NTEKPC) were the most frequent. Our analysis allowed us to identify 25 different NTEKPC, mainly belonging to the NTEKPC-I, and a new type (proposed as IVa) was also observed. This is the first systematic review that consolidates information about the behavior of the blaKPC acquisition in P. aeruginosa and the genetic platforms implied in its successful worldwide spread. Our results show high NTEKPC prevalence in P. aeruginosa and an accelerated dynamic of unrelated clones. All information collected in this review was used to build an interactive online map.

Within patient genetic diversity of blaKPC harboring Klebsiella pneumoniae in a Colombian hospital and identification of a new NTEKPC platform.

Resistance to carbapenems in Klebsiella pneumoniae has been mostly related with the worldwide dissemination of KPC, largely due to the pandemic clones belonging to the complex clonal (CC) 258. To unravel blaKPC post-endemic clinical impact, here we describe clinical characteristics of 68 patients from a high complexity hospital, and the molecular and genetic characteristics of their 139 blaKPC-K. pneumoniae (KPC-Kp) isolates. Of the 26 patients that presented relapses or reinfections, 16 had changes in the resistance profiles of the isolates recovered from the recurrent episodes. In respect to the genetic diversity of KPC-Kp isolates, PFGE revealed 45 different clonal complexes (CC). MLST for 12 representative clones showed ST258 was present in the most frequent CC (23.0%), however, remaining 11 representative clones belonged to non-CC258 STs (77.0%). Interestingly, 16 patients presented within-patient genetic diversity of KPC-Kp clones. In one of these, three unrelated KPC-Kp clones (ST258, ST504, and ST846) and a blaKPC-K. variicola isolate (ST182) were identified. For this patient, complete genome sequence of one representative isolate of each clone was determined. In K. pneumoniae isolates blaKPC was mobilized by two Tn3-like unrelated platforms: Tn4401b (ST258) and Tn6454 (ST504 and ST846), a new NTEKPC-IIe transposon for first time characterized also determined in the K. variicola isolate of this study. Genome analysis showed these transposons were harbored in different unrelated but previously reported plasmids and in the chromosome of a K. pneumoniae (for Tn4401b). In conclusion, in the blaKPC post-endemic dissemination in Colombia, different KPC-Kp clones (mostly non-CC258) have emerged due to integration of the single blaKPC gene in new genetic platforms. This work also shows the intra-patient resistant and genetic diversity of KPC-Kp isolates. This circulation dynamic could impact the effectiveness of long-term treatments.

Extracellular vesicles of U937 macrophage cell line infected with DENV-2 induce activation in endothelial cells EA.hy926.

Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.

First Report and Comparative Genomics Analysis of a blaOXA-244-Harboring Escherichia coli Isolate Recovered in the American Continent.

The carbapenemase OXA-244 is a derivate of OXA-48, and its detection is very difficult in laboratories. Here, we report the identification and genomic analysis of an Escherichia coli isolate (28Eco12) harboring the blaOXA-244 gene identified in Colombia, South America. The 28Eco12 isolate was identified during a retrospective study, and it was recovered from a patient treated in Colombia. The complete nucleotide sequence was established using the PacBio platform. A comparative genomics analysis with other blaOXA-244-harboring Escherichia coli strains was performed. The 28Eco12 isolate belonged to sequence type (ST) 38, and its genome was composed of two molecules, a chromosome of 5,343,367 bp and a plasmid of 92,027 bp, which belonged to the incompatibility group IncY and did not harbor resistance genes. The blaOXA-244 gene was chromosomally encoded and mobilized by an ISR1-related Tn6237 composite transposon. Notably, this transposon was inserted and located within a new genomic island. To our knowledge, this is the first report of a blaOXA-244-harboring Escherichia coli isolate in America. Our results suggest that the introduction of the OXA-244-producing E. coli isolate was through clonal expansion of the ST38 pandemic clone. Other isolates producing OXA-244 could be circulating silently in America.

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone.

BACKGROUND: Pseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia. RESULTS: In a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241 bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2″)-Ia, two Tn402-like with ant(3″)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background. CONCLUSION: This is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

Genomic Epidemiology of NDM-1-Encoding Plasmids in Latin American Clinical Isolates Reveals Insights into the Evolution of Multidrug Resistance.

Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-ß-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.

Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300

ABSTRACT Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300.

Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

Emergence and spread of a new community-genotype methicillin-resistant Staphylococcus aureus clone in Colombia.

BACKGROUND: Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) clones are a global concern due to their resistance and increased virulence and their ability to cause infections both hospitalized patients and healthy people in the community. Here, we characterize 32 isolates of a new CG-MRSA clone. These isolates were identified in four cities in Colombia, South America. METHODS: The isolates were recovered from four different epidemiological and prospective studies that were conducted in several regions of Colombia. Molecular characterizations included multilocus sequence typing; pulsed-field gel electrophoresis; SCCmec, agr and spa typing; and whole-genome sequencing. RESULTS: All isolates belonged to ST923 (clonal complex 8), harbouring SCCmec IVa and a spa type t1635 and lacking an arginine catabolism mobile element. The isolates were classified as COL923, were resistant to at least one non-beta-lactam antibiotic, and exhibited high frequencies (>60%) of resistance to macrolides and tetracycline. Using whole-genome sequencing, we found that this new clone harbours novel prophage 3 and beta-island structures and a slightly different pathogenicity island 5. Moreover, isolates belonging to the COL923 clone are grouped in a different clade than USA300 and USA300-LV. CONCLUSION: Our results show the emergence and spread of the COL923 clone in different cities in Colombia. This clone is resistant to several antibiotics and possesses new structures in its mobile genetic elements.

[Methicillin-sensitive Staphylococcus aureus isolates related to USA300 clone: Origin of community-genotype MRSA in Colombia?]. / Aislamientos de Staphylococcus aureus sensibles a meticilina relacionados genéticamente con el clon USA300, ¿origen de los aislamientos SARM de genotipo comunitario en Colombia?

INTRODUCTION: USA300 is a genetic lineage found both in methicillin-resistant (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolates. In Colombia, hospital and community MRSA infections are caused by a USA300-related community genotype MRSA (CG-MRSA) clone. The genetic origin of this clone is unknown yet. OBJECTIVE: To identify and characterize methicillin-resistant (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates in order to improve the information about the origin of the CG-MRSA isolates in Colombia. MATERIALS AND METHODS: USA300-related MSSA isolates were detected and characterized from a study of 184 S. aureus isolates (90 MRSA and 94 MSSA) recovered from infections. The genetic relatedness of the isolates was established by means of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and protein A gene typification ( spa typing). RESULTS: Among 184 isolates, 27 (14.7%) showed molecular characteristics and genetic relationship with the USA300 clone, of which 18 were MRSA and nine were MSSA. All USA300-related MRSA harbored Staphylococcal cassette chromosome mec (SCC mec ) IVc (3.1.2). In the MSSA isolates, SCC mec remnants or att B duplicate sites were not detected. CONCLUSIONS: In Colombia, the CG-MRSA isolates probably originated in the dissemination of an USA300-related MSSA clone which later acquired SCC mec IVc.

USA300-related methicillin-resistant Staphylococcus aureus clone is the predominant cause of community and hospital MRSA infections in Colombian children.

OBJECTIVE: Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) isolates are known to be more virulent and clinically aggressive in children. The goal of the present study was characterize the molecular epidemiology of MRSA isolates causing infections in Colombian children. METHODS: An observational and prospective study was conducted between April 2009 and June 2011 at 15 hospitals in Bogotá, Colombia. A detailed epidemiological profile was made of 162 children infected with MRSA. The isolates were subjected to antimicrobial susceptibility testing, molecular characterization including 21 virulence genes, SCCmec, spa and agr typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). RESULTS: Among all isolates included in the study, 85.8% were obtained from patients whose infectious process was initiated in the community; of these, 69,8% occurred in patients without healthcare-associated risk factors. The molecular characterization of the isolates showed a high proportion (95.1%) containing a community-genotype profile with a high prevalence of SCCmec type IV, PVL-positives, and also related to CC8. Most CG-MRSA isolates (143, 92.9%) were genetically related to the pandemic clone USA300, differing by the presence of SCCmec IVc and the absence of the arginine catabolic mobile element (ACME). CONCLUSIONS: An increase in the frequency of CG-MRSA infections has been reported worldwide. In this study we found that almost all MRSA infections in our pediatric population were caused by community-genotype isolates, supporting the success of the CG-MRSA clones.

Aislamientos de Staphylococcus aureus sensibles a meticilina relacionados genéticamente con el clon USA300, ¿origen de los aislamientos SARM de genotipo comunitario en Colombia? / Methicillin-sensitive Staphylococcus aureus isolates related to USA300 clone: Origin of community-genotype MRSA in Colombia?

Introducción. USA300 es un linaje genético que se encuentra en aislamientos de Staphylococcus aureus sensibles (SASM) y resistentes a meticilina (SARM). Actualmente, en Colombia las infecciones por SARM en hospitales y en la comunidad son causadas principalmente por un clon con genotipo comunitario (SARM-GC) relacionado genéticamente con el clon USA300. El origen de esta variante es aún desconocido. Objetivo. Identificar y caracterizar aislamientos de S. aureus resistentes y sensibles a meticilina con el fin de aportar información para establecer un posible origen de los aislamientos SARM-GC en Colombia. Materiales y métodos. Se realizó una caracterización de aislamientos SASM relacionados con el clon USA300 detectados a partir de un análisis de 184 aislamientos de S. aureus (90 SARM y 94 SASM) causantes de infecciones. La relación genética de los aislamientos se determinó por electroforesis en gel de campo pulsado (PFGE), tipificación de secuencias multilocus (MLST) y tipificación del gen de la proteína A ( spa ). Resultados. De los 184 aislamientos, 27 (14,7 %) presentaron características moleculares y relación genética con el clon USA300, y de ellos, 18 fueron SARM y nueve fueron SASM. Todos los aislamientos SARM relacionados con este clon albergaban un casete estafilocócico cromosómico mec (SCC mec ) IVc (3.1.2). En ningún aislamiento SASM se detectaron secuencias remanentes de SCC mec o una duplicación del sitio att B que evidenciaran la pérdida del casete. Conclusión. El origen de los aislamientos SARM-GC en Colombia probablemente se encuentre en la diseminación de clones SASM relacionados con el clon USA300 que adquirieron el SCC mec IVc posteriormente.

Introduction: USA300 is a genetic lineage found both in methicillin-resistant (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolates. In Colombia, hospital and community MRSA infections are caused by a USA300-related community genotype MRSA (CG-MRSA) clone. The genetic origin of this clone is unknown yet. Objective: To identify and characterize methicillin-resistant (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates in order to improve the information about the origin of the CG-MRSA isolates in Colombia. Materials and methods: USA300-related MSSA isolates were detected and characterized from a study of 184 S. aureus isolates (90 MRSA and 94 MSSA) recovered from infections. The genetic relatedness of the isolates was established by means of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and protein A gene typification ( spa typing). Results: Among 184 isolates, 27 (14.7%) showed molecular characteristics and genetic relationship with the USA300 clone, of which 18 were MRSA and nine were MSSA. All USA300-related MRSA harbored Staphylococcal cassette chromosome mec (SCC mec ) IVc (3.1.2). In the MSSA isolates, SCC mec remnants or att B duplicate sites were not detected. Conclusions: In Colombia, the CG-MRSA isolates probably originated in the dissemination of an USA300-related MSSA clone which later acquired SCC mec IVc.

Outbreak of NDM-1-producing Klebsiella pneumoniae in a neonatal unit in Colombia.

Six multiresistant, NDM-1-producing Klebsiella pneumoniae strains were recovered from an outbreak that affected six neonatal patients in a Colombian hospital. Molecular analysis showed that all of the isolates harbored the blaNDM-1, qnrA, and intI1 genes and were clonally related. Multilocus sequence typing showed that the isolates belonged to a new sequence type (ST1043) that was different from the sequence types that had previously been reported. This is the first report of NDM-1-producing isolates in South America.

Detection of a new community genotype methicillin-resistant Staphylococcus aureus clone that is unrelated to the USA300 clone and that causes pediatric infections in Colombia.

The dissemination of a clone of community genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) that is related to USA300 has been reported in Latin America. We recently detected isolates of a new clone of CG-MRSA (spa type t1635 and ACME-negative) that was genetically unrelated to the USA300 clone and that causes infections in children in Colombia. This finding indicates the appearance of a new clone of CG-MRSA in our region.