Javier Escobar-Perez

Carbapenem-resistant Pseudomonas aeruginosa is a major cause of healthcare associated infections in hospitalized patients and what is more warring with reduced therapeutic options. The KPC is a powerful enzyme capable of hydrolyzing the carbapenems, described first in Klebsiella pneumoniae and it already has found in P. aeruginosa.Objective: To perform a comparative genomic analysis of two new genetic platforms mobilizing the bla_KPC-2 and bla_KPC-3 in two ST111 and ST235 pandemic clones of P. aeruginosa in Colombia, South America. Methods: Sixty-six bla_KPC-harboring P. aeruginosa isolates were identified and characterized during a prospective study conducted in six high complex hospitals in Colombia. Genetic platforms mobilizing the bla_KPC were analyzed. Results: The bla_KPC-2 and bla_KPC-3 were identified in 24 and 42 isolates, respectively. The bla_KPC-2-harboring isolates belonged to ST235 and bla_KPC-3 to ST111. The whole genome sequencing indicated that the bla_KPC-3 gene was mobilized by the Tn4401b within a 55-kb-size environmental origin plasmid, which, in other isolates, was inserted into the chromosome through a transposition event of ISPa38. Regarding the bla_KPC-2 gene, this was mobilized by a new Non-Tn4401 Element (NTE) derived from transposon Tn2 (proposed as variant IIg), which has been transposed into a 43-Kb-size little-studied plasmid related to Klebsiella spp. Conclusions: Our results reveal a new acquisition event of bla_KPC in P. aeruginosa, in this case bla_KPC-3. Likewise, the pandemic high-risk clones ST111 and ST235 of P. aeruginosa continues to spread bla_KPC gene through different mobile genetic elements, jumping of conventional Tn4401b and acquiring new Tn2-derived NTE, which were inserted in diverse plasmids.

The Com_YlbF domain-containing proteins inhibit sporulation, competition, and biofilm formation by affecting the activity of ribonuclease RNase-Y in Bacillus subtilis . Similar Com_YlbF proteins are found in Staphylococcus aureus , but their function is yet to be determined. This study investigates the role of Com_YlbF domain-containing proteins (Qrp/YheA, YmcA, and YlbF) in S. aureus by evaluating the impact of qrp/yheA , ymcA , and ylbF gene deletion on biofilm formation, PIA/PNAG production, and hemolytic capacity. It was found that deletion of the three qrp/yheA , ymcA , and ylbF genes in S. aureus produced a decrease in biofilm formation, a slight decrease in PIA/PNAG production, and a decrease in its hemolytic capacity. Global transcriptional analysis in the mutant strain showed dysregulation of several genes associated with pathogenesis, most notably a decreased expression of the agrA gene (quorum sensing system), the delta , alpha , and gamma hemolysins, as well as the sdrC genes and eap/map (adhesion proteins) genes. It also showed an attenuation of virulence, manifesting as enhanced survival among Galleria mellonella larvae and BALB/c mice, possibly associated due to disrupted RNase-Y activity when the qrp/yheA , ymcA , and ylbF are deleted. In conclusion, this work supports the hypothesis that this new family of proteins containing a Com_YlbF domain is involved in the regulation of genes related to biofilm formation, hemolysis, and virulence in S. aureus through a change in RNase-Y activity. IMPORTANCE: The Com_YlbF proteins are a group of small proteins identified in some Gram-positive bacteria whose function has not been described, but whose deletion has been related to pleiotropic effects in Bacillus subtilis (reduction of biofilm ability, sporulation, and competence). However, the role of these proteins in S. aureus has been little studied. This study shows the importance of the Com_YlbF protein family in S. aureus involved in biofilm formation and hemolysis. The phenotypes could be explained by an alteration of the enzymatic activity of the endoribonuclease RNase-Y, resulting in reduced secretion of virulence factors. These findings represent a significant step forward in understanding the modulation of pathogenesis and virulence in S. aureus .

The com_ylbF domain-containing proteins that inhibit sporulation, competition, and biofilm formation by affecting the activity of Ribonuclease RNase-Y in Bacillus subtilis . Similar Com_YlbF proteins are found in Staphylococcus aureus , but their function is yet to be determined. This study investigates the role of com_ylbF domain-containing proteins (Qrp/YheA, YmcA, and YlbF) in S. aureus by evaluating the impact of qrp/yheA , ymcA, and ylbF gene deletion on biofilm formation, PIA/PNAG production, and hemolytic capacity. It was found that deletion of the three qrp/yheA , ymcA, and ylbF genes in S. aureus produced a decrease in biofilm formation, a slight decrease in PIA/PNAG production, and a decrease in its hemolytic capacity. Global transcriptional analysis in the mutant strain showed dysregulation of several genes associated with pathogenesis, notably a decreased expression of the agrA gene (quorum sensing system), the delta , alpha, and gamma hemolysins, as well as the sdrC genes, eap/map (adhesion proteins). It also showed-an attenuation of virulence, manifesting as enhanced survival among Galleria mellonella larvae, and BALB/c mice, possibly associated due to disrupted RNase-Y activity when the qrp/yheA , ymcA, and ylbF are deleted. In conclusion, this work supports the hypothesis that this new family of proteins containing a com_ylbF domain is involved in the regulation of genes related to biofilm formation, hemolysis, and virulence in S. aureus through a change in RNase-Y activity.

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 bla _KPC-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 bla _KPC-2 in Pseudomonas aeruginosa .

The role ofBlastocystisin 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 byBlastocystisin 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 ofBlastocystisin 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 byBlastocystis. Regarding taxonomic characterization, SpA patients colonized byBlastocystisshowed significant increases in the phylumPseudomonadota, classGammaproteobacteria, familySuccinivibrionaceae, and genusSuccinivibrio. Simultaneously, there were significant increases in the classBacilli, orderLactobacillales, familiesLactobacillaceaeandClostridiaceae,and generaLactobacillusandClostridiumin non-colonized SpA patients. On the other hand, PICRUSt analysis inBlastocystis-positive SpA patients showed elevations in pathways that may enhance antioxidant capacities and alleviate intestinal inflammation, whileBlastocystis-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.

The dissemination of bla_KPC-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 bla_KPC genetic environment. We found 691 KPC-Pa isolates belonging to 41 different sequence types and recovered from 14 countries. Although the bla_KPC gene is still mobilized by the transposon Tn4401, the non-Tn4401 elements (NTE_KPC) were the most frequent. Our analysis allowed us to identify 25 different NTE_KPC, mainly belonging to the NTE_KPC-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 bla_KPC acquisition in P. aeruginosa and the genetic platforms implied in its successful worldwide spread. Our results show high NTE_KPC 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.

The multi-resistance presented by P. aeruginosa has greatly increased due to the presence of genes for carbapenemases such as bla_KPC. The dissemination of this gene has been associated with the Tn4401, the main mobile genetic element that carries bla_KPC in its structure. However, some non-Tn4401 elements (NTE_KPC) associated with bla_KPC have been found in different bacteria. Here, we characterized in silico and in vitro bla_KPC-associated elements in P. aeruginosa. To identify these elements, a search algorithm was performed using NCBI databases; sequences were filtered and pair-aligned to describe the bla_KPC genetic environment. Additionally, a PCR-based strategy was designed to target Tn4401 variants and NTE_KPC groups and assessed in 61 Colombian clinical isolates. Using an in silico approach, 51 bla_KPC-positive entries longer than 3kb (in the bla_KPC upstream region) were found; from these, 72.7% carried an NTE_KPC. On the PCR assay, Tn4401 was the most frequent element among the P. aeruginosa in Colombia. However, NTE_KPC-IIf was presented on 29.5% of the isolates, in different genetic lineages and at least in four hospitals. These results show high NTE_KPC prevalence in P. aeruginosa.

Staphylococcus aureus and Staphylococcus epidermidis have microbial surface components recognizing adhesive matrix molecules (MSCRAMM) adhesion proteins that enhance their biofilm formation ability, as well as virulence factors that influence morbidity and mortality in hospital settings. In this work, four peptides analogous of the peptide LL-37 that were evaluated to inhibit biofilm formation and its action potential on the expression of MSCRAMM proteins in clinical isolates through different tests, such as crystal violet, PCR and qPCR. In total, 96.8% of S. aureus were strong in biofilm formation in contrast to 48.4% of S. epidermidis. sdrG and sdrF genes were present in 100% of S. epidermidis strains and in all isolates. In S. aureus, specific genes that code for MSCRAMM proteins were detected: clfA (89%), clFB, sdrC and fnBA (94%). The peptides did not show hemolytic or cytotoxic activity. In this study, it was evidenced that of the peptides DLL37-1 at a 5 µM concentration was an efficacious antimicrobial agent and depicted greater biofilm inhibition in both bacterial species. Exhibiting a significant inhibition rate in S. aureus, this peptide caused a negative regulation in the expression of the genes clfA and sdrC, showed greater biological activity.