Liquid Fungal Cocultivation as a Strategy to Access Bioactive Metabolites.

Fungi are a rich source of bioactive compounds. Fungal cocultivation is a method of potentiating chemical interactions and, consequently, increasing bioactive molecule production. In this study, we evaluated the bactericidal, antiprotozoal, and cathepsin V inhibition activities of extracts from axenic cultures of 6 fungi (Fusarium guttiforme, Pestalotiopsis diospyri, Phoma caricae-papayae, Colletotrichum horii, Phytophthora palmivora, and C. gloeosporioides) that infest tropical fruits and 57 extracts obtained by their cocultivation. Our results reveal that fungal cocultivation enhances the biological activity of the samples, since all extracts that were active on Gram-positive bacteria, Gram-negative bacteria, Trypanosoma cruzi, and Leishmania infantum were obtained from cocultivation. Bacterial growth is either totally or partially inhibited by 46% of the extracts. Two extracts containing mainly fusaric and 9,10-dehydrofusaric acids were particularly active. The presence of the fungus F. guttiforme in co-cultures that give rise to extracts with the highest activities against L. infantum. An axenic culture gave rise to the most active extract for the inhibition of cathepsin V; however, other coculture extracts also exhibited activity toward this biological target. Therefore, the results of the biological activities indicate that fungal cocultivation increased the biological potential of samples, likely due to the hostile and competitive environment that pushes microorganisms to produce substances important for defense and allows access to metabolic routes then silenced in milder cultivation conditions.

Dissemination of blaKPC-2 in an NTEKPC by an IncX5 plasmid.

blaKPC-2 is disseminated worldwide usually in Tn4401, a Tn3-family transposon, and primarily in Klebsiella pneumoniae ST258, a well-known lineage that is distributed worldwide and responsible for several outbreaks. Although occurring rarely, blaKPC-2 has been described in non-Tn4401 elements (NTEKPCs), first in China and then in a few other countries. This study reports the dissemination of a blaKPC-2-carrying NTEKPC among ST11/CG258 K. pneumoniae strains and ST1642 K. quasipneumoniae subsp. quasipneumoniae AMKP9 in an Amazonian hospital. The dissemination was due to pAMKP10, an ~48 kbp IncX5 plasmid carrying ΔISKpn6/blaKPC-2/ISKpn27 in a Tn1722-based unit. Although similar to NTEKPC-Ia from pKP048 described in China, a different transposase is present upstream of ISKpn27. Additionally, mutations were identified downstream of ISKpn27 but did not affect the blaKPC-2 promoter regions. pAMKP10 conjugated in vitro only from CG258 isolates. Since CG258 strains are generally well adapted to the hospital environment, it is significant that pAMKP10 has found its way into this clinically significant clonal group. The impact of inter- and intraspecies dissemination of NTEKPCs and IncX5 plasmids harboring carbapenem resistance genes is unknown, but monitoring these plasmids could reveal their dissemination preferences.

Oxidative functionalization of a halimane diterpenoid achieved by fungal transformation.

Regio and stereoselective activation of sp3 CH bonds remain one of the major advantages of biocatalysis over traditional chemocatalytic methods. Herein, we describe the oxy-functionalization of halimane diterpenoid 1 by whole cells of three filamentous fungi, aiming to obtain derivatives with desirable biological properties. After incubating 1 with Fusarium oxysporum, Myrothecium verrucaria, and Rhinocladiella similis at different concentrations and incubation times, four known (3, 5, 6, and 7) and three new (2, 4, and 8) halimane derivatives were obtained and characterized. F. oxysporum catalyzed the hydroxylation of positions C-2 (2) and C-7 (4), while R. similis simultaneously mediated the 2-oxo-functionalization and the hydration of 13,14-(CC)double bond belonging to an α,ß-unsaturated carbonyl system (8). Compounds 1-7 were non-cytotoxic against HCT-116 and MCF-7 cancer cell lines at tested concentrations. However, substrate 1 displayed moderate reduction ability against biofilm produced by Staphylococcus epidermidis ATCC35984 (84% at 1.6 mM), and this effect was retained to some extent by derivatives 4 and 7. These results emphasize the prominent potential of filamentous fungi associated with the microbiota of medicinal plants as versatile catalysts for singularly useful reactions through their complex enzymatic machinery, as well as the high susceptibility of halimane-diterpenoid substrates.

Optimizing Bothropstoxin-I-Derived Peptides: Exploring the Antibacterial Potential of p-BthW.

Antimicrobial peptides are an emerging class of antibiotics that present a series of advantageous characteristics such as wide structural variety, broad spectrum of activity, and low propensity to select for resistance. They are found in all classes of life as defense molecules. A group of peptides derived from the protein Bothropstoxin-I has been previously studied as an alternative treatment against multi-drug-resistant bacteria. The peptide p-BthTX-I (sequence: KKYRYHLKPFCKK) and its homodimer, linked by disulfide oxidation through the residues of Cys11 and the serum degradation product [sequence: (KKYRYHLKPFC)2], were evaluated and showed similar antimicrobial activity. In this study, we synthesized an analogue of p-BthTX-I that uses the strategy of Fmoc-Lys(Fmoc)-OH in the C-terminal region for dimerization and tryptophan for all aromatic amino acids to provide better membrane interactions. This analogue, named p-BthW, displayed potent antibacterial activity at lower concentrations and maintained the same hemolytic levels as the original molecule. Our assessment revealed that p-BthW has a quick in vitro bactericidal action and prolonged post-antibiotic effect, comparable to the action of polymyxin B. The mode of action of p-BthW seems to rely not only on membrane depolarization but also on necrosis-like effects, especially in Gram-negative bacteria. Overall, the remarkable results regarding the propensity to develop resistance reaffirmed the great potential of the developed molecule.

Antimicrobial Activity of an Fmoc-Plantaricin 149 Derivative Peptide against Multidrug-Resistant Bacteria.

Antimicrobial resistance poses a major threat to public health. Given the paucity of novel antimicrobials to treat resistant infections, the emergence of multidrug-resistant bacteria renewed interest in antimicrobial peptides as potential therapeutics. This study designed a new analog of the antimicrobial peptide Plantaricin 149 (Pln149-PEP20) based on previous Fmoc-peptides. The minimal inhibitory concentrations of Pln149-PEP20 were determined for 60 bacteria of different species and resistance profiles, ranging from 1 mg/L to 128 mg/L for Gram-positive bacteria and 16 to 512 mg/L for Gram-negative. Furthermore, Pln149-PEP20 demonstrated excellent bactericidal activity within one hour. To determine the propensity to develop resistance to Pln149-PEP20, a directed-evolution in vitro experiment was performed. Whole-genome sequencing of selected mutants with increased MICs and wild-type isolates revealed that most mutations were concentrated in genes associated with membrane metabolism, indicating the most likely target of Pln149-PEP20. Synchrotron radiation circular dichroism showed how this molecule disturbs the membranes, suggesting a carpet mode of interaction. Membrane depolarization and transmission electron microscopy assays supported these two hypotheses, although a secondary intracellular mechanism of action is possible. The molecule studied in this research has the potential to be used as a novel antimicrobial therapy, although further modifications and optimization remain possible.

Polymyxin Resistance Among XDR ST1 Carbapenem-Resistant Acinetobacter baumannii Clone Expanding in a Teaching Hospital.

Acinetobacter baumannii is an opportunistic pathogen primarily associated with multidrug-resistant nosocomial infections, for which polymyxins are the last-resort antibiotics. This study investigated carbapenem-resistant A. baumannii strains exhibiting an extensively drug-resistant (XDR) phenotype, including four isolates considered locally pan drug-resistant (LPDR), isolated from inpatients during an outbreak at a teaching hospital in Brazil. ApaI DNA macrorestriction followed by PFGE clustered the strains in three pulsotypes, named A to C, among carbapenem-resistant A. baumannii strains. Pulsotypes A and B clustered six polymyxin-resistant A. baumannii strains. MLST analysis of representative strains of pulsotypes A, B, and C showed that they belong, respectively, to sequence types ST1 (clonal complex, CC1), ST79 (CC79), and ST903. Genomic analysis of international clones ST1 and ST79 representative strains predicted a wide resistome for ß-lactams, aminoglycosides, fluoroquinolones, and trimethoprim-sulfamethoxazole, with bla OXA-23 and bla OXA-72 genes encoding carbapenem resistance. Amino acid substitutions in PmrB (Thr232Ile or Pro170Leu) and PmrC (Arg125His) were responsible for polymyxin resistance. Although colistin MICs were all high (MIC ≥ 128 mg/L), polymyxin B MICs varied; strains with Pro170Leu substitution in PmrB had MICs > 128 mg/L, while those with Thr232Ile had lower MICs (16-64 mg/L), irrespective of the clone. Although the first identified polymyxin-resistant A. baumannii strain belonged to ST79, the ST1 strains were endemic and caused the outbreak most likely due to polymyxin B use. The genome comparison of two ST1 strains from the same patient, but one susceptible and the other resistant to polymyxin, revealed mutations in 28 ORFs in addition to pmrBC. The ORF codifying an acyl-CoA dehydrogenase has gained attention due to its fatty acid breakdown and membrane fluidity involvement. However, the role of these mutations in the polymyxin resistance mechanism remains unknown. To prevent the dissemination of XDR bacteria, the hospital infection control committee implemented the patient bathing practice with a 2% chlorhexidine solution, a higher concentration than all A. baumannii chlorhexidine MICs. In conclusion, we showed the emergence of polymyxin resistance due to mutations in the chromosome of the carbapenem-resistant A. baumannii ST1, a high-risk global clone spreading in this hospital.

An RpoB mutation confers dual heteroresistance to daptomycin and vancomycin in Staphylococcus aureus.

We have previously reported the establishment of a Staphylococcus aureus laboratory strain, 10 3d1, having reduced susceptibility to daptomycin and heterogeneous vancomycin-intermediate S. aureus (VISA) phenotype. The strain was generated in vitro by serial daptomycin selection (Camargo, I. L., H. M. Neoh, L. Cui, and K. Hiramatsu, Antimicrob. Agents Chemother. 52:4289-4299, 2008). Here we explored the genetic mechanism of resistance in the strain by whole-genome sequencing and by producing gene-replaced strains. By genome comparison between 10 3d1 and its parent methicillin-resistant Staphylococcus aureus (MRSA) strain N315ΔIP, we identified five nonsynonymous single nucleotide polymorphisms (SNPs). One of the five mutations was found in the rpoB gene encoding the RNA polymerase ß subunit. The mutation at nucleotide position 1862 substituted the 621st alanine by glutamic acid. The replacement of the intact rpoB with the mutated rpoB, designated rpoB(A621E), conferred N315ΔIP with the phenotypes of reduced susceptibility to daptomycin and hetero-VISA. The rpoB(A621E)-mediated resistance conversion was accompanied by a thickened cell wall and reduction of the cell surface negative charge. Being consistent with these phenotypic changes, microarray data showed that the expression of the dlt operon, which increases the cell surface positive charge, was enhanced in the rpoB(A621E) mutant. Other remarkable findings of microarray analysis of the rpoB(A621E) mutant included repression of metabolic pathways of purine, pyrimidine, arginine, the urea cycle, and the lac operon, enhancement of the biosynthetic pathway of vitamin B2, K1, and K2, and cell wall metabolism. Finally, mutations identified in rplV and rplC, encoding 50S ribosomal proteins L22 and L3, respectively, were found to be associated with the slow growth, but not with the phenotype of decreased susceptibility to vancomycin and daptomycin, of 10 3d1.

Molecular basis for the emergence of a new hospital endemic tigecycline-resistant Enterococcus faecalis ST103 lineage.

Enterococcus faecalis are a major cause of nosocomial infection worldwide, and the spread of vancomycin resistant strains (VRE) limits treatment options. Tigecycline-resistant VRE began to be isolated from inpatients at a Brazilian hospital within months following the addition of tigecycline to the hospital formulary. This was found to be the result of a spread of an ST103 E. faecalis clone. Our objective was to identify the basis for tigecycline resistance in this lineage. The genomes of two closely related tigecycline-susceptible (MIC = 0.06 mg/L), and three representative tigecycline-resistant (MIC = 1 mg/L) ST103 isolates were sequenced and compared. Further, efforts were undertaken to recapitulate the emergence of resistant strains in vitro. The specific mutations identified in clinical isolates in several cases were within the same genes identified in laboratory-evolved strains. The contribution of various polymorphisms to the resistance phenotype was assessed by trans-complementation of the wild type or mutant alleles, by testing for differences in mRNA abundance, and/or by examining the phenotype of transposon insertion mutants. Among tigecycline-resistant clinical isolates, five genes contained non-synonymous mutations, including two genes known to be related to enterococcal tigecycline resistance (tetM and rpsJ). Finally, within the in vitro-selected resistant variants, mutation in the gene for a MarR-family response regulator was associated with tigecycline resistance. This study shows that E. faecalis mutates to attain tigecycline resistance through the complex interplay of multiple mechanisms, along multiple evolutionary trajectories.

Serial daptomycin selection generates daptomycin-nonsusceptible Staphylococcus aureus strains with a heterogeneous vancomycin-intermediate phenotype.

In order to better understand the mechanism of daptomycin resistance, we generated a daptomycin-nonsusceptible derivative strain, strain 10*3d1 (MIC = 3.0 microg/ml), by in vitro exposure of methicillin-resistant Staphylococcus aureus strain N315DeltaIP (MIC = 0.5 microg/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 10*3d1-10 (MIC = 1.0 microg/ml), by passaging 10*3d1 in drug-free medium for 10 days. The resultant triple-isogenic strains were analyzed for their phenotypes and gene expression by microarray analysis. No significant differences in the membrane fluidities of 10*3d1 and 10*3d1-10 compared to the membrane fluidity of N315DeltaIP were observed. Resistant strain 10*3d1 had the highest membrane potential, followed by strains 10*3d1-10 and N315DeltaIP. The vancomycin and teicoplanin MICs also increased. Teichoic acid genes (tagA, tagG), mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were downregulated in 10*3d1 and 10*3d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 10*3d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 10*3d1 was significantly thicker than that of the parental strain. Taken together, daptomycin exposure selected a daptomycin-nonsusceptible strain with a phenotype similar to that of heterogeneous vancomycin-intermediate S. aureus and a transcription profile that partially overlapped that of heterogeneous vancomycin-intermediate S. aureus.

Daptomycin and vancomycin heteroresistance revealed among CC5-SCCmecII MRSA clone and in vitro evaluation of treatment alternatives.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to the success of clinical treatment. Besides high antimicrobial resistance rates, the presence of heterogeneous vancomycin-intermediate S. aureus (hVISA) and heterogeneous daptomycin-non-susceptible S. aureus (hDNSSA) in the hospital environment is underestimated and is associated with treatment failure. The aim of this study was to investigate MRSA dissemination in a Brazilian hospital and to evaluate the efficacy of various treatment options in vitro. METHODS: MRSA strains were typed by MLST, PFGE and SCCmec typing. Minimum inhibitory concentrations (MICs) to daptomycin, linezolid, quinupristin/dalfopristin, teicoplanin, tetracycline, tigecycline, vancomycin and tedizolid were determined by broth microdilution. The presence of a heterogeneous population was detected by population analysis profile (PAP). Regarding hVISA and hDNSSA strains, the sequences and expression levels of genes involved in resistance to daptomycin and vancomycin were determined as well as cell wall thickness and autolysis. RESULTS: ST5/ST105-SCCmecII lineage was prevalent amongst 27 clinical MRSA characterised in this study. Two hDNSSA strains (one also hVISA) were detected and were confirmed by PAP. Isolate SCMSC29 (hVISA and hDNSSA) showed increased expression of genes involved in cell wall metabolism, slight cell wall thickening, reduction of autolysis, and single nucleotide polymorphisms (SNPs) in the rpoB and mprF genes compared with the susceptible strain SCMSC31. SCMSC35 (hDNSSA) presented SNPs in the rpoB and mprF genes as well as a thickened cell wall. CONCLUSIONS: Despite this worrying and hard to detect phenotype, treatment alternatives such as teicoplanin, linezolid, tetracycline, tigecycline, quinupristin/dalfopristin and tedizolid were all active against these isolates.

Molecular analysis of methicillin-resistant Staphylococcus aureus dissemination among healthcare professionals and/or HIV patients from a tertiary hospital.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen in community settings. MRSA colonized individuals may contribute to its dissemination; the risk of MRSA infection is increased in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, although the prevalence of colonization in this group is not well established. The present study addressed this issue by characterizing MRSA isolates from HIV/AIDS patients and their healthcare providers (HCPs) to determine whether transmission occurred between these two populations. METHODS: A total of 24 MRSA isolates from HIV-infected patients and five from HCPs were collected between August 2011 and May 2013. Susceptibility to currently available antimicrobials was determined. Epidemiological typing was carried out by pulsed-field gel electrophoresis, multilocus sequence typing, and Staphylococcus cassette chromosome (SCCmec) typing. The presence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and heterogeneous daptomycin-resistant Staphylococcus aureus (hDRSA) was confirmed by population analysis profile. Isolates characterized in this study were also compared to isolates from 2009 obtained from patients at the same hospital. RESULTS: A variety of lineages were found among patients, including ST5-SCCmecII and ST30-SCCmecIV. Two isolates were Panton-Valentine leukocidin-positive, and hVISA and hDRSA were detected. MRSA isolates from two HCPs were not related to those from HIV/AIDS patients, but clustered with archived MRSA from 2009 with no known relationship to the current study population. CONCLUSIONS: ST105-SCCmecII clones that colonized professionals in 2011 and 2012 were already circulating among patients in 2009, but there is no evidence that these clones spread to or between HIV/AIDS patients up to the 7th day of their hospitalization.

Molecular analysis of methicillin-resistant Staphylococcus aureus dissemination among healthcare professionals and/or HIV patients from a tertiary hospital

Abstract INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen in community settings. MRSA colonized individuals may contribute to its dissemination; the risk of MRSA infection is increased in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, although the prevalence of colonization in this group is not well established. The present study addressed this issue by characterizing MRSA isolates from HIV/AIDS patients and their healthcare providers (HCPs) to determine whether transmission occurred between these two populations. METHODS: A total of 24 MRSA isolates from HIV-infected patients and five from HCPs were collected between August 2011 and May 2013. Susceptibility to currently available antimicrobials was determined. Epidemiological typing was carried out by pulsed-field gel electrophoresis, multilocus sequence typing, and Staphylococcus cassette chromosome (SCCmec) typing. The presence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and heterogeneous daptomycin-resistant Staphylococcus aureus (hDRSA) was confirmed by population analysis profile. Isolates characterized in this study were also compared to isolates from 2009 obtained from patients at the same hospital. RESULTS: A variety of lineages were found among patients, including ST5-SCCmecII and ST30-SCCmecIV. Two isolates were Panton-Valentine leukocidin-positive, and hVISA and hDRSA were detected. MRSA isolates from two HCPs were not related to those from HIV/AIDS patients, but clustered with archived MRSA from 2009 with no known relationship to the current study population. CONCLUSIONS: ST105-SCCmecII clones that colonized professionals in 2011 and 2012 were already circulating among patients in 2009, but there is no evidence that these clones spread to or between HIV/AIDS patients up to the 7th day of their hospitalization.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice.

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.