Phenotypic and molecular characterization of Staphylococcus aureus isolates conducted in nares of psoriatic patients attending a public hospital in Argentina / Caracterización fenotípica y molecular de aislamientos de Staphylococcus aureus portados en narinas de pacientes con psoriasis que concurren a un hospital público en Argentina

Abstract Although Staphylococcus aureus increases its relative abundance in psoriasis when compared with the microbiome of healthy subjects, it is not the most important microorganism underlying this disease. However, there is scant data on the role and molecular features of S. aureus strains in psoriasis; therefore, the aim of this study was to evaluate nasal carriage of this microorganism, its phenotypic and molecular characteristics as well as the impact of host factors on its carriage in psoriatic patients. The presence of S. aureus was analyzed in nasal swabs from 46 healthy volunteers and 50 psoriatic patients by conventional microbiology techniques. Nasal carriage of S. aureus was higher in psoriatic patients than in the control group (37.24% vs 22.98%, respectively), being associated to sex (male), age (adults) and severity of the disease (more frequent in moderate and severe cases). Determination of antibiotic resistance detected 12% of (-lactam resistant isolates, with variable accompanying resistance to macrolides, aminoglycosides and fluoroquinolones. No resistance to rifampicin, vancomycin, mupirocin or trimethoprim/sulfamethoxazole was found. A preliminary molecular characterization of the isolates was performed by PCR amplification of virulence genes. Molecular characterization of the strains did not reveal a predominant strain in psoriatic patients. Although we established host factors related to increased carriage of S. aureus in psoriatic patients, we could not establish the predominance of one type of strain. Genomic and transcriptomic analysis of the isolated strains would be necessary to address this point.

Resumen A pesar de que Staphylococcus aureus incrementa su abundancia relativa en la psoriasis cuando se compara con el microbioma de personas sanas, no es el microorganismo más importante subyacente a la enfermedad. Sin embargo, existen pocos datos sobre el papel y las características moleculares de las cepas de S. aureus en pacientes con psoriasis. Nuestro objetivo fue evaluar la portación nasal de este microorganismo, sus características fenotípicas y moleculares, y el impacto de factores del hospedador sobre dicha portación en estos pacientes. Se analizó la presencia de S. aureus en hisopados nasales de 46 voluntarios sanos y 50 pacientes con psoriasis mediante técnicas microbiológicas convencionales. Se encontró mayor portación en pacientes con psoriasis que en el grupo control (37,24% vs. 22,98%, respectivamente) y esta estuvo asociada al sexo (masculino), la edad (adultos) y la gravedad de la enfermedad (más frecuente en casos moderados a graves). El 12% de los aislamientos de S. aureus mostraron resistencia a betalactámicos, con resistencia acompañante a macrólidos, aminoglucósidos y fluoroquinolonas en grado variable. No se encontró resistencia a rifampicina, vancomicina, mupirocina o trimetroprima/sulfametoxazol. Se realizó una caracterización molecular preliminar de los aislamientos por amplificación de genes de virulencia mediante PCR. Si bien se identificaron factores relacionados con el hospedador que incrementan la portación nasal de S. aureus en pacientes con psoriasis, la caracterización molecular de las cepas no reveló ninguna característica genotípica predominante asociada a esta afección. Se necesitan más estudios genómicos y transcriptómicos para profundizar en esta caracterización.

Phenotypic and molecular characterization of Staphylococcus aureus isolates conducted in nares of psoriatic patients attending a public hospital in Argentina.

Although Staphylococcus aureus increases its relative abundance in psoriasis when compared with the microbiome of healthy subjects, it is not the most important microorganism underlying this disease. However, there is scant data on the role and molecular features of S. aureus strains in psoriasis; therefore, the aim of this study was to evaluate nasal carriage of this microorganism, its phenotypic and molecular characteristics as well as the impact of host factors on its carriage in psoriatic patients. The presence of S. aureus was analyzed in nasal swabs from 46 healthy volunteers and 50 psoriatic patients by conventional microbiology techniques. Nasal carriage of S. aureus was higher in psoriatic patients than in the control group (37.24% vs 22.98%, respectively), being associated to sex (male), age (adults) and severity of the disease (more frequent in moderate and severe cases). Determination of antibiotic resistance detected 12% of ß-lactam resistant isolates, with variable accompanying resistance to macrolides, aminoglycosides and fluoroquinolones. No resistance to rifampicin, vancomycin, mupirocin or trimethoprim/sulfamethoxazole was found. A preliminary molecular characterization of the isolates was performed by PCR amplification of virulence genes. Molecular characterization of the strains did not reveal a predominant strain in psoriatic patients. Although we established host factors related to increased carriage of S. aureus in psoriatic patients, we could not establish the predominance of one type of strain. Genomic and transcriptomic analysis of the isolated strains would be necessary to address this point.

Bioinformatic Analysis of a Set of 14 Temperate Bacteriophages Isolated from Staphylococcus aureus Strains Highlights Their Massive Genetic Diversity.

Epidemiology and virulence studies of Staphylococcus aureus showed that temperate bacteriophages are one of the most powerful drivers for its evolution not only because of their abundance but also because of the richness of their genetic payload. Here, we report the isolation, genome sequencing, and bioinformatic analysis of 14 bacteriophages induced from lysogenic S. aureus strains from human or veterinary (cattle) origin. The bacteriophages belonged to the Siphoviridae family; were of similar genome size (40 to 45 kbp); and fell into clusters B2, B3, B5, and B7 according to a recent clustering proposal. One of the phages, namely, vB_SauS_308, was the most unusual one, belonging to the sparsely populated subcluster B7 but showing differences in protein family contents compared with the rest of the members. This phage contains a type I endolysin (one catalytic domain and noncanonical cell wall domain [CBD]) and a host recognition module lacking receptor binding protein, cell wall hydrolase, and tail fiber proteins. This phage also lacked virulence genes, which is opposite to what has been reported for subcluster B6 and B7 members. None of six phages, taken as representatives of each of the four subclusters, showed activity on coagulase-negative staphylococci (excepted for two Staphylococcus hominis strains in which propagation and a very slow adsorption rate were observed) nor transducing ability. Immunity tests on S. aureus RN4220 lysogens with each of these phages showed no cross immunity. IMPORTANCE To the best of our knowledge, this set of sequenced bacteriophages is the largest one in South America. Our report describes for the first time the utilization of MultiTwin software to analyze the relationship between phage protein families. Notwithstanding the fact that most of the genetic information obtained correlated with recently published information, due to their geographical origin, the reported analysis adds up to and confirms currently available knowledge of Staphylococcus aureus temperate bacteriophages in terms of phylogeny and role in host evolution.

Unveiling the Biosynthetic Pathway for Short Mycolic Acids in Nontuberculous Mycobacteria: Mycobacterium smegmatis MSMEG_4301 and Its Ortholog Mycobacterium abscessus MAB_1915 Are Essential for the Synthesis of α'-Mycolic Acids.

Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.

New one-pot synthesis of anti-tuberculosis compounds inspired on isoniazid.

A library of thirty N-substituted tosyl N'-acryl-hydrazones was prepared with p-toluenesulfonyl hydrazide, methyl propiolate and different aldehydes in a one-pot synthesis via an aza-Michael reaction. The scope of the reaction was studied, including aliphatic, isoprenylic, aromatic and carbocyclic aldehydes. The prepared collection was tested against Mycobacterium tuberculosis H37Rv. Nine analogs of the collection showed Minimum Inhibitory Concentration ≤10 µM, of which the most active members (MIC of 1.25 µM) were exclusively E isomers. In order to validate the mechanism of action of the most active acrylates, we tested their activity on a M. tuberculosis InhA over-expressing strain obtaining MIC that consistently doubled those obtained on the wild type strain. Additionally, the binding mode of those analogs on M. tuberculosis InhA was investigated by docking simulations. The results displayed a hydrogen bond interaction between the sulfonamide and Ile194 and the carbonyl of the methyl ester with Tyr 158 (both critical residues in the interaction with the fatty acyl chain substrate), where the main differences on the binding mode relays on the hydrophobicity of the nitrogen substituent. Additionally, chemoinformatic analysis was performed to evaluate in silico possible cytotoxicity risk and ADME-Tox profile. Based on their simple preparation and interesting antimycobacterial activity profile, the newly prepared aza-acrylates are promising candidates for antitubercular drug development.

Mutations in the anti-sigma H factor RshA confer resistance to econazole and clotrimazole in Mycobacterium smegmatis.

Azole drugs such as econazole, are active on Mycobacterium tuberculosis and Mycobacterium smegmatis ; however, the identification of their target(s) is still pending. It has been reported that mutations in the non-essential system mmpL5-mmpS5 conferred resistance to econazole in M. tuberculosis . We herein report that an azole-resistant mutant screen in M. smegmatis rendered mutations in rshA, encoding a non-essential anti-sigma H protein.

Deletion of MSMEG_1350 in Mycobacterium smegmatis causes loss of epoxy-mycolic acids, fitness alteration at low temperature and resistance to a set of mycobacteriophages.

Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.

A katG S315T or an ahpC promoter mutation mediate Mycobacterium tuberculosis resistance to 2-thiophen carboxylic acid hydrazide, an inhibitor resembling the anti-tubercular drugs Isoniazid and Ethionamide.

Clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis are differentially susceptible to 2-Thiophen Hydrazide (TCH); however its mechanism of action or the reasons for that difference are unknown. We report herein that under our experimental conditions, TCH inhibits M. tuberculosis in solid but not in liquid medium, and that in spite of resembling Isoniazid and Ethionamide, it does not affect mycolic acid synthesis. To understand the mechanisms of action of TCH we isolated M. tuberculosis TCH resistant mutants which fell into two groups; one resistant to TCH and Isoniazid but not to Ethionamide or Triclosan, and the other resistant only to TCH with no, or marginal, cross resistance to Isoniazid. A S315T katG mutation conferred resistance to TCH while katG expression from a plasmid reduced M. tuberculosis MIC to this drug, suggesting a possible involvement of KatG in TCH activation. Whole genome sequencing of mutants from this second group revealed a single mutation in the alkylhydroperoxide reductase ahpC promoter locus in half of the mutants, while the remaining contained mutations in dispensable genes. This is the first report of the genetics underlying the action of TCH and of the involvement of ahpC as the sole basis for resistance to an anti-tubercular compound.

Pre-clinical evaluation of quinoxaline-derived chalcones in tuberculosis.

New effective compounds for tuberculosis treatment are needed. This study evaluated the effects of a series of quinoxaline-derived chalcones against laboratorial strains and clinical isolates of M. tuberculosis. Six molecules, namely N5, N9, N10, N15, N16, and N23 inhibited the growth of the M. tuberculosis H37Rv laboratorial strain. The three compounds (N9, N15 and N23) with the lowest MIC values were further tested against clinical isolates and laboratory strains with mutations in katG or inhA genes. From these data, N9 was selected as the lead compound for further investigation. Importantly, this chalcone displayed a synergistic effect when combined with moxifloxacin. Noteworthy, the anti-tubercular effects of N9 did not rely on inhibition of mycolic acids synthesis, circumventing important mechanisms of resistance. Interactions with cytochrome P450 isoforms and toxic effects were assessed in silico and in vitro. The chalcone N9 was not predicted to elicit any mutagenic, genotoxic, irritant, or reproductive effects, according to in silico analysis. Additionally, N9 did not cause mutagenicity or genotoxicity, as revealed by Salmonella/microsome and alkaline comet assays, respectively. Moreover, N9 did not inhibit the cytochrome P450 isoforms CYP3A4/5, CYP2C9, and CYP2C19. N9 can be considered a potential lead molecule for development of a new anti-tubercular therapeutic agent.

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis.

The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741-1743 (highly homologous to a Pseudomonas aeruginosa acyl-CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886-MSMEG_1741-1743 strictly required oleic acid for growth. The ΔMSMEG_1886-ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742-43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.

Broad-range lytic bacteriophages that kill Staphylococcus aureus local field strains.

Staphylococcus aureus is a very successful opportunistic pathogen capable of causing a variety of diseases ranging from mild skin infections to life-threatening sepsis, meningitis and pneumonia. Its ability to display numerous virulence mechanisms matches its skill to display resistance to several antibiotics, including ß-lactams, underscoring the fact that new anti-S. aureus drugs are urgently required. In this scenario, the utilization of lytic bacteriophages that kill bacteria in a genus -or even species- specific way, has become an attractive field of study. In this report, we describe the isolation, characterization and sequencing of phages capable of killing S. aureus including methicillin resistant (MRSA) and multi-drug resistant S. aureus local strains from environmental, animal and human origin. Genome sequencing and bio-informatics analysis showed the absence of genes encoding virulence factors, toxins or antibiotic resistance determinants. Of note, there was a high similarity between our set of phages to others described in the literature such as phage K. Considering that reported phages were obtained in different continents, it seems plausible that there is a commonality of genetic features that are needed for optimum, broad host range anti-staphylococcal activity of these related phages. Importantly, the high activity and broad host range of one of our phages underscores its promising value to control the presence of S. aureus in fomites, industry and hospital environments and eventually on animal and human skin. The development of a cocktail of the reported lytic phages active against S. aureus-currently under way- is thus, a sensible strategy against this pathogen.

Antitubercular activity of 1,2,3-triazolyl fatty acid derivatives.

A collection of 1,2,3-triazoles unsaturated fatty acid mimics were efficiently synthesized by click chemistry. The 1,4-disubstituted analogs prepared covered different alkyl chain lengths and triazole positions. The compounds were subsequently tested against Mycobacterium tuberculosis, being most of them active with some of the analogs displaying activity at micromolar concentration. The most potent member of the series has the triazole moiety on the C-2 position with a carbon chain of eight or ten carbon atoms. The 1,5-isomers of the most active analog were significantly less active than the original isomer. The activity of the selected hit was assayed on several clinical MTB multi-drug resistant strains providing the same MIC.

Anti-tubercular and antioxidant activities of C-glycosyl carbonic anhydrase inhibitors: towards the development of novel chemotherapeutic agents against Mycobacterium tuberculosis.

During the treatment of tuberculosis infection, oxidative stress due to anti-tubercular drugs may result in tissue inflammation. It was suggested that treatment with antioxidant drugs could be beneficial as an adjunct to anti-tuberculosis drug therapy. Recently our group has shown that several C-glycosides are inhibitors of Mycobacterium tuberculosis ß-carbonic anhydrases (CAs, EC 4.2.1.1). In an effort to develop novel chemotherapeutic agents against tuberculosis, the anti-tubercular and antioxidant activities of a series of C-glycosides containing the phenol or the methoxyaryl moiety were studied. Many compounds showed inhibition of growth of M. tuberculosis H37Rv strain and good antioxidant ability. A glycomimetic incorporating the 3-hydroxyphenyl moiety showed the best activity profile and therefore this functionality represents lead for the development of novel anti-tubercular agents with dual mechanisms of action.

Recycling and refurbishing old antitubercular drugs: the encouraging case of inhibitors of mycolic acid biosynthesis.

One of the first approaches undertaken in the quest for antitubercular compounds was that of understanding the mechanism of action of old drugs and proposing chemical modifications or other strategies to improve their activity, generally lost to the mechanisms of resistance developed by Mycobacterium tuberculosis. A leading case was the work carried out on a set of compounds with proven activity on the essential pathway of the synthesis of mycolic acids. As a result, different solutions were presented, improving the activity of those inhibitors or producing novel compounds acting on the same molecular target(s), but avoiding the most common resistance strategies developed by the tubercle bacilli. This review focuses on the activity of those compounds, developed following the completion of the studies on several of the classic antitubercular drugs.

Analysis of novel mycobacteriophages indicates the existence of different strategies for phage inheritance in mycobacteria.

Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

Virulence factors of the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world.

Inhibition of the ß-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: identification of the first inhibitor with anti-mycobacterial activity.

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis ß-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H(37)Rv strain, leading to the identification of (E)-1-(2',3',4',6'-tetra-O-acetyl-ß-D-glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.

Mutations in the essential FAS II ß-hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii.

It has recently been shown that the anti-mycobacterial pro-drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build-up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that 'de novo' biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC-resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti-mycobacterial compound.