Effects of Dimerization, Dendrimerization, and Chirality in p-BthTX-I Peptide Analogs on the Antibacterial Activity and Enzymatic Inhibition of the SARS-CoV-2 PLpro Protein.

Recent studies have shown that the peptide [des-Cys11,Lys12,Lys13-(p-BthTX-I)2K] (p-Bth) is a p-BthTX-I analog that shows enhanced antimicrobial activity, stability and hemolytic activity, and is easy to obtain compared to the wild-type sequence. This molecule also inhibits SARS-CoV-2 viral infection in Vero cells, acting on SARS-CoV-2 PLpro enzymatic activity. Thus, the present study aimed to assess the effects of structural modifications to p-Bth, such as dimerization, dendrimerization and chirality, on the antibacterial activity and inhibitory properties of PLpro. The results showed that the dimerization or dendrimerization of p-Bth was essential for antibacterial activity, as the monomeric structure led to a total loss of, or significant reduction in, bacterial activities. The dimers and tetramers obtained using branched lysine proved to be prominent compounds with antibacterial activity against Gram-positive and Gram-negative bacteria. In addition, hemolysis rates were below 10% at the corresponding concentrations. Conversely, the inhibitory activity of the PLpro of SARS-CoV-2 was similar in the monomeric, dimeric and tetrameric forms of p-Bth. Our findings indicate the importance of the dimerization and dendrimerization of this important class of antimicrobial peptides, which shows great potential for antimicrobial and antiviral drug-discovery campaigns.

Synthesis and Antibacterial Activity of Polyalthic Acid Analogs.

Polyalthic acid (PA) is a diterpene found in copaiba oil. As a continuation of our work with PA, we synthesized PA analogs and investigated their antibacterial effects on preformed biofilms of Staphylococcus epidermidis and determined the minimal inhibitory concentration (MIC) of the best analogs against planktonic bacterial cells. There was no difference in activity between the amides 2a and 2b and their corresponding amines 3a and 3b regarding their ability to eradicate biofilm. PA analogs 2a and 3a were able to significantly eradicate the preformed biofilm of S. epidermidis and were active against all the Gram-positive bacteria tested (Enterococcus faecalis, Enterococcus faecium, S. epidermidis, Staphylococcus aureus), with different MIC depending on the microorganism. Therefore, PA analogs 2a and 3a are of interest for further in vitro and in vivo testing to develop formulations for antibiotic drugs against Gram-positive bacteria.

Characterization of blaKPC-2 and blaNDM-1 Plasmids of a K. pneumoniae ST11 Outbreak Clone.

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring blaKPC and blaNDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including blaKPC-2 and blaNDM-1. The blaKPC gene was present on a ~56 Kbp IncN plasmid and the blaNDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the blaNDM plasmid contained genes for conjugational transfer, only the blaKPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the blaKPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The blaKPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the blaNDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored blaKPC-2 and blaNDM-1. The blaKPC-harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the blaKPC-containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil.

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Resistance in In Vitro Selected Tigecycline-Resistant Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Is Driven by Mutations in mepR and mepA Genes.

A tigecycline-susceptible (TGC-S) Sequence Type (ST) 5 clinical methicillin-resistant Staphylococcus aureus (MRSA) strain was cultured in escalating levels of tigecycline, yielding mutants eightfold more resistant. Their genomes were sequenced to identify genetic alterations, resulting in resistance. Alterations in rpsJ, commonly related to tigecycline resistance, were also investigated. Tigecycline resistance was mediated by loss-of-function mutations in the transcriptional repressor mepR, resulting in derepression of the efflux pump mepA. Increased levels of resistance were obtained by successive mutations in mepA itself. No alterations in RpsJ were observed in selected strains, but we observed a K57M substitution, previously correlated with resistance, among TGC-S clinical strains. Thus, the pathway to tigecycline resistance in CC5 MRSA in vitro appears to be derepression of mep operon as the result of mepR loss-of-function mutation, followed by alterations in MepA efflux pump. This shows that other evolutionary pathways, besides mutation of rpsJ, are available for evolving tigecycline resistance in CC5 MRSA.

Different VanA Elements in E. faecalis and in E. faecium Suggest at Least Two Origins of Tn1546 Among VRE in a Brazilian Hospital.

In 2009 during surveillance in a Brazilian hospital, many patients were confirmed to be colonized by vancomycin-resistant enterococci (VRE) and a few infection cases occurred. Among 14 isolates of Enterococcus faecalis, most had the same pulsotype, virulence profile (agg(+)elrA(+)gelE(+)), and were sequence type (ST)103, indicating dissemination of a clone. The 47 Enterococcus faecium were separated into four pulsotypes, the predominant virulence profile being esp(+)acm(+). All of them harbored the hospital marker IS16, and three randomly chosen isolates were ST412, belonging to the Clonal Complex 17. E. faecalis were all susceptible to penicillin and ampicillin, while all E. faecium were resistant to them. All isolates were susceptible to daptomycin and tigecycline. There were no rep-family genes common to all VRE. The VanA element of all E. faecium lost its left-side inverted repeat (IRL) region and had a specific IS insertion. On the other hand, all E. faecalis presented intact Tn1546. The size of plasmids containing the vanA gene as well as its rep-families varied between and within species. The lack of a vanA plasmid common to all VRE, together with the differences among VanA elements, despite the fact that some patients were colonized by both species during their hospitalization, leads us to suggest at least two different Tn1546 origins.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.