Drug treatments and vaccine designs against the opportunistic human pathogen Pseudomonas aeruginosa have multiple issues, all associated with the diverse genetic traits present in this pathogen, ranging from multi-drug resistant genes to the molecular machinery for the biosynthesis of biofilms. Several candidate vaccines against P. aeruginosa have been developed, which target the outer membrane proteins; however, major issues arise when attempting to establish complete protection against this pathogen due to its presumably genotypic variation at the strain level. To shed light on this concern, we proposed this study to assess the P. aeruginosa pangenome and its molecular evolution across multiple strains. The P. aeruginosa pangenome was estimated to contain more than 16,000 non-redundant genes, and approximately 15 % of these constituted the core genome. Functional analyses of the accessory genome indicated a wide presence of genetic elements directly associated with pathogenicity. An in-depth molecular evolution analysis revealed the full landscape of selection forces acting on the P. aeruginosa pangenome, in which purifying selection drives evolution in the genome of this human pathogen. We also detected distinctive positive selection in a wide variety of outer membrane proteins, with the data supporting the concept of substantial genetic variation in proteins probably recognized as antigens. Approaching the evolutionary information of genes under extremely positive selection, we designed a new Multi-Locus Sequencing Typing assay for an informative, rapid, and cost-effective genotyping of P. aeruginosa clinical isolates. We report the unprecedented pangenome characterization of P. aeruginosa on a large scale, which included almost 200 bacterial genomes from one single species and a molecular evolutionary analysis at the pangenome scale. Evolutionary information presented here provides a clear explanation of the issues associated with the use of protein conjugates from pili, flagella, or secretion systems as antigens for vaccine design, which exhibit high genetic variation in terms of non-synonymous substitutions in P. aeruginosa strains.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of bla _KPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of bla _KPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015).

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with an increase in the frequency of infections caused by multidrug resistant (MDR) and extensively drug resistant (XDR) strains, limiting the available therapeutic options. The most troublesome resistance is the acquisition and production of carbapenemases such as Verona integron-encoded metallo-β-lactamases (VIM), the most frequent and widespread, and the Klebsiella pneumoniae carbapenemases (KPC), which has continuously spread in the last decade. Its dissemination is linked to their location on mobile genetic elements (MGEs). In Colombia, VIM and KPC have been increasing in its frequency showing major successful dissemination. In this article, we molecularly characterized and analyzed the genetic context of bla_VIM and bla_KPC in carbapenem-resistant P. aeruginosa (CRPA) isolates from infected and colonized patients in two tertiary-care hospitals, one in Medellín and the other in a municipality close to Medellín, both areas with high carbapenemase endemicity in Colombia (2013–2015). Using whole-genome sequencing (WGS), we identified a remarkable variety of genetic backgrounds in these MDR P. aeruginosa isolates carrying bla_KPC–2 and bla_VIM–2. There were a diversity of class 1 integron and variations in the gene cassettes associated to bla_VIM–2, as well as a possible event of spread of bla_KPC–2 mediated by a plasmid that contained part of Tn4401b in one infection case. The dissemination of bla_VIM–2 and bla_KPC–2 in P. aeruginosa in this area in Colombia has been strongly influenced by successful international clones, carrying these genes and additional determinants of resistance on MGEs, accompanied by gene rearrangement under an antimicrobial selection pressure. These findings emphasize the need to implement control strategies based on rational antibiotic use.

An Escherichia coli isolate sequence-type 471 (ST471) producing Verona integron-encoded metallo-β-lactamases (VIM)-4 was recovered from a rectal swab in a patient without travel records with osteomyelitis in Colombia. The isolate carried a class 1 integron-borne bla _VIM-4 gene with a 170-bp duplication in the 3′ end of the gene, preceded by an aac(6 ′)-Ib gene. The genetic environment of bla _VIM-4 , bla _CMY-2 , and sul2 genes showed similarities to the backbone of pKKp4, an IncA/C-type plasmid from a Klebsiella pneumoniae strain carrying bla _VIM-4 recovered in Kuwait. This is the first report of bla _VIM-4 in Enterobacterales in South America. Our results suggest that bla _VIM-4 gene was found on an IncA/C-type plasmid that could play a role in the spread of VIM-4 carbapenemase in Colombia.

Background: Drug treatments and vaccine designs against the opportunistic human pathogen Pseudomonas aeruginosa have multiple issues, all associated with the diverse genetic traits present in this pathogen, ranging from multi-drug resistant genes to the molecular machinery for the biosynthesis of biofilms. Several candidate vaccines against P. aeruginosa have been developed, which target the outer membrane proteins; however, major issues arise when attempting to establish complete protection against this pathogen due to its presumably genotypic variation at the strain level. To shed light on this concern, we proposed this study to assess the P. aeruginosa pangenome and its molecular evolution across multiple strains. Results: The P. aeruginosa pangenome was estimated to contain more than 16,000 non-redundant genes, and approximately 15% of these constituted the core genome. Functional analyses of the accessory genome indicated a wide presence of genetic elements directly associated with pathogenicity. An in-depth molecular evolution analysis revealed the full landscape of selection forces acting on the P. aeruginosa pangenome, in which purifying selection drives evolution in the genome of this human pathogen. We also detected distinctive positive selection in a wide variety of outer membrane proteins, with the data supporting the concept of substantial genetic variation in proteins probably recognized as antigens. Approaching the evolutionary information of genes under extremely positive selection, we designed a new Multi-Locus Sequencing Typing assay for an informative, rapid, and cost-effective genotyping of P. aeruginosa clinical isolates. Conclusions: We report the unprecedented pangenome characterization of P. aeruginosa on a large scale, which included almost 200 bacterial genomes from one single species and a molecular evolutionary analysis at the pangenome scale. Evolutionary information presented here provides a clear explanation of the issues associated with the use of protein conjugates from pili, flagella, or secretion systems as antigens for vaccine design, which exhibit high genetic variation in terms of non-synonymous substitutions in P. aeruginosa strains.

Introduction. The search for microorganisms producing antimicrobial compounds repre sents a strategy to address the global crisis of antibiotic resistance. Identification of bacteria of the genus Streptomyces with antagonistic activity against resistant bacteria is the initial step for the subsequent recovery of effective antibacterial metabolites for the inhibition of these resistant organisms. Objectives. Recognize promising sources to isolate filamentous bacteria able to inhibit clinical isolates, including ESBL- and KPC- producing Entero bacteriaceae and MRSA. Methods. Actinobacteria were isolated from rhizospheres and a compost system. MRSA, Escherichia coli, and Klebsiella pneumoniae clinical isolates were chosen to address antagonism tests. Antibacterial activity was recorded by a cross-streak method. Streptomyces isolates were characterized according to International Streptomyces Project and the native isolates with antimicrobial activity were identified by molecular te chniques. Results. Nine isolates of actinobacteria with activity against resistant-antibiotic bacteria were obtained, two from the avocado rhizosphere, four from a living fence and three from a composting system. Two of the isolates showed activity against all the tested antibiotic-resistant bacteria. Molecular taxonomic identification found S. jumonjinensis, S. bacillaris, S. prasinus, S. microflavus, and S. cadmiisoli as putative species for native iso lates. Conclusions. Streptomyces with antibacterial activities against ESBL and KPC-pro ducing Enterobacteria and MRSA have been isolated and the potential of rhizospheres and compost systems for obtaining antibiotic-producing bacteria was validated. Native isolates exhibited common traits for Streptomyces; although NCBI’s Blast did not show a resolution to identification, EzBioCloud 16S-based identification was able to accurately detect the identity of the isolates down to the species level.