Novel Variant and Known Mutation in 23S rRNA Gene of Mycoplasma pneumoniae, Northern Vietnam, 2023.

During a 2023 outbreak of Mycoplasma pneumoniae-associated community-acquired pneumonia among children in northern Vietnam, we analyzed M. pneumoniae isolated from nasopharyngeal samples. In almost half (6 of 13) of samples tested, we found known A2063G mutations (macrolide resistance) and a novel C2353T variant on the 23S rRNA gene.

Biocompatible Ti3Au-Ag/Cu thin film coatings with enhanced mechanical and antimicrobial functionality.

BACKGROUND: Biofilm formation on medical device surfaces is a persistent problem that shelters bacteria and encourages infections and implant rejection. One promising approach to tackle this problem is to coat the medical device with an antimicrobial material. In this work, for the first time, we impart antimicrobial functionality to Ti3Au intermetallic alloy thin film coatings, while maintaining their superior mechanical hardness and biocompatibility. METHODS: A mosaic Ti sputtering target is developed to dope controlled amounts of antimicrobial elements of Ag and Cu into a Ti3Au coating matrix by precise control of individual target power levels. The resulting Ti3Au-Ag/Cu thin film coatings are then systematically characterised for their structural, chemical, morphological, mechanical, corrosion, biocompatibility-cytotoxicity and antimicrobial properties. RESULTS: X-ray diffraction patterns reveal the formation of a super hard ß-Ti3Au phase, but the thin films undergo a transition in crystal orientation from (200) to (211) with increasing Ag concentration, whereas introduction of Cu brings no observable changes in crystal orientation. Scanning and transmission electron microscopy analysis show the polyhedral shape of the Ti3Au crystal but agglomeration of Ag particles between crystal grains begins at 1.2 at% Ag and develops into large granules with increasing Ag concentration up to 4.1 at%. The smallest doping concentration of 0.2 at% Ag raises the hardness of the thin film to 14.7 GPa, a 360% improvement compared to the ∼4 GPa hardness of the standard Ti6Al4V base alloy. On the other hand, addition of Cu brings a 315-330% improvement in mechanical hardness of films throughout the entire concentration range of 0.5-7.1 at%. The thin films also show good electrochemical corrosion resistance and a > tenfold reduction in wear rate compared to Ti6Al4V alloy. All thin film samples exhibit very safe cytotoxic profiles towards L929 mouse fibroblast cells when analysed with Alamar blue assay, with ion leaching concentrations lower than 0.2 ppm for Ag and 0.08 ppm for Cu and conductivity tests reveal the positive effect of increased conductivity on myogenic differentiation. Antimicrobial tests show a drastic reduction in microbial survival over a short test period of < 20 min for Ti3Au films doped with Ag or Cu concentrations as low as 0.2-0.5 at%. CONCLUSION: Therefore, according to these results, this work presents a new antimicrobial Ti3Au-Ag/Cu coating material with excellent mechanical performance with the potential to develop wear resistant medical implant devices with resistance to biofilm formation and bacterial infection.

Phylogenomic Reappraisal of Fatty Acid Biosynthesis, Mycolic Acid Biosynthesis and Clinical Relevance Among Members of the Genus Corynebacterium.

The genus Corynebacterium encompasses many species of biotechnological, medical or veterinary significance. An important characteristic of this genus is the presence of mycolic acids in their cell envelopes, which form the basis of a protective outer membrane (mycomembrane). Mycolic acids in the cell envelope of Mycobacterium tuberculosis have been associated with virulence. In this study, we have analysed the genomes of 140 corynebacterial strains, including representatives of 126 different species. More than 50% of these strains were isolated from clinical material from humans or animals, highlighting the true scale of pathogenic potential within the genus. Phylogenomically, these species are very diverse and have been organised into 19 groups and 30 singleton strains. We find that a substantial number of corynebacteria lack FAS-I, i.e., have no capability for de novo fatty acid biosynthesis and must obtain fatty acids from their habitat; this appears to explain the well-known lipophilic phenotype of some species. In most species, key genes associated with the condensation and maturation of mycolic acids are present, consistent with the reports of mycolic acids in their species descriptions. Conversely, species reported to lack mycolic acids lacked these key genes. Interestingly, Corynebacterium ciconiae, which is reported to lack mycolic acids, appears to possess all genes required for mycolic acid biosynthesis. We suggest that although a mycolic acid-based mycomembrane is widely considered to be the target for interventions by the immune system and chemotherapeutics, the structure is not essential in corynebacteria and is not a prerequisite for pathogenicity or colonisation of animal hosts.

The influence of linkages between 1-hydroxy-2(1H)-pyridinone coordinating groups and a tris(2-aminoethyl)amine core in a novel series of synthetic hexadentate iron(III) chelators on antimicrobial activity.

Resistance of pathogens to antimicrobials is a major current healthcare concern. In a series of linked studies, we have investigated synthetic iron chelators based on hydroxy-pyridinone ligands as novel bacteriostatic agents. Herein we describe our synthesis of several useful building blocks based on the 1-hydroxy-2(1H)-pyridinone moiety, including a novel formyl derivative, which were combined with a tris(2-aminoethyl)amine core to obtain a series of new high-affinity hexadentate Fe(III) chelators. The design principle examined by this series is the size and flexibility of the linker between the core and the metal ligands. Measurement of the pKa and stability constants (Fe3+ and Cu2+) of representative coordinating groups was performed to help rationalise the biological activity of the chelators. The novel chelators were tested on a panel of representative microorganisms with some effectively inhibiting microbial growth. We demonstrate that the nature and position of the linker between the hydroxypyridinone and the tris(2-aminoethyl)amine core has considerable impact upon microbial growth inhibition and that both amide or amine linkages can give efficacious chelators.

Genomic analysis of a novel Rhodococcus (Prescottella) equi isolate from a bovine host.

Rhodococcus (Prescottella) equi causes pneumonia-like infections in foals with high mortality rates and can also infect a number of other animals. R. equi is also emerging as an opportunistic human pathogen. In this study, we have sequenced the genome of a novel R. equi isolate, B0269, isolated from the faeces of a bovine host. Comparative genomic analyses with seven other published R. equi genomes, including those from equine or human sources, revealed a pangenome comprising of 6876 genes with 4141 genes in the core genome. Two hundred and 75 genes were specific to the bovine isolate, mostly encoding hypothetical proteins of unknown function. However, these genes include four copies of terA and five copies of terD genes that may be involved in responding to chemical stress. Virulence characteristics in R. equi are associated with the presence of large plasmids carrying a pathogenicity island, including genes from the vap multigene family. A BLAST search of the protein sequences from known virulence-associated plasmids (pVAPA, pVAPB and pVAPN) revealed a similar plasmid backbone on two contigs in bovine isolate B0269; however, no homologues of the main virulence-associated genes, vapA, vapB or vapN, were identified. In summary, this study confirms that R. equi genomes are highly conserved and reports the presence of an apparently novel plasmid in the bovine isolate B0269 that needs further characterisation to understand its potential involvement in virulence properties.

Synthesis of novel Iron(III) chelators based on triaza macrocycle backbone and 1-hydroxy-2(H)-pyridin-2-one coordinating groups and their evaluation as antimicrobial agents.

Several novel chelators based on 1-hydroxy-2(1H)-pyridinone coordinating groups decorating a triaza macrocyclic backbone scaffold were synthesised as potential powerful Fe(3+) chelators capable of competing with bacterial siderophores. In particular, a novel chloromethyl derivative of 1-hydroxy-2(1H)-pyridinone exploiting a novel protective group for this family of coordinating groups was developed. These are the first examples of hexadentate chelators based on 1-hydroxy-2(1H)-pyridinone to be shown to have a biostatic activity against a range of pathogenic bacteria. Their efficacy as biostatic agents was assessed revealing that minor variations in the structure of the chelator can affect efficacy profoundly. The minimal inhibitory concentrations of our best tested novel chelators approach or are comparable to those for 1,4,7-tris(3-hydroxy-6-methyl-2-pyridylmethyl)-1,4,7-triazacyclononane, the best Fe(3+) chelator known to date. The retarding effect these chelators have on microbial growth suggests that they could have a potential application as a co-active alongside antibiotics in the fight against infections.

Antibacterial Metallic Touch Surfaces.

Our aim is to present a comprehensive review of the development of modern antibacterial metallic materials as touch surfaces in healthcare settings. Initially we compare Japanese, European and US standards for the assessment of antimicrobial activity. The variations in methodologies defined in these standards are highlighted. Our review will also cover the most relevant factors that define the antimicrobial performance of metals, namely, the effect of humidity, material geometry, chemistry, physical properties and oxidation of the material. The state of the art in contact-killing materials will be described. Finally, the effect of cleaning products, including disinfectants, on the antimicrobial performance, either by direct contact or by altering the touch surface chemistry on which the microbes attach, will be discussed. We offer our outlook, identifying research areas that require further development and an overview of potential future directions of this exciting field.

Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi.

Virulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel ß-barrel fold with ß1-ß2-ß3-ß8-ß5-ß6-ß7-ß4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the ß-barrel. Possible functional significance of these recent structures is discussed.

Sterol 3ß-glucosyltransferase biocatalysts with a range of selectivities, including selectivity for testosterone.

The main objectives of this work were to characterise a range of purified recombinant sterol 3ß-glucosyltransferases and show that rational sampling of the diversity that exists within sterol 3ß-glucosyltransferase sequence space can result in a range of enzyme selectivities. In our study the catalytically active domain of the Saccharomyces cerevisiae 3ß-glucosyltransferase was used to mine putative sterol 3ß-glucosyltransferases from the databases. Selected diverse sequences were expressed in and purified from Escherichia coli and shown to have different selectivities for the 3ß-hydroxysteroids ergosterol and cholesterol. Surprisingly, three enzymes were also selective for testosterone, a 17ß-hydroxysteroid. This study therefore reports for the first time sterol 3ß-glucosyltransferases with selectivity for both 3ß- and 17ß-hydroxysteroids and is also the first report of recombinant 3ß-glucosyltransferases with selectivity for steroids with a hydroxyl group at positions other than C-3. These enzymes could therefore find utility in the pharmaceutical industry for the green synthesis of a range of glycosylated compounds of medicinal interest.

Genome sequence of the Fleming strain of Micrococcus luteus, a simple free-living actinobacterium.

Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.

Lipoteichoic acid biosynthesis: two steps forwards, one step sideways?

Lipoteichoic acids (LTAs) are membrane-anchored molecules in the cell envelopes of Gram-positive bacteria. Until recently, they were considered to be restricted to the Firmicutes, which include important pathogens such as Staphylococcus aureus and Streptococcus pneumoniae. Polyanionic LTAs have fundamentally important roles in divalent cation retention within the Gram-positive cell envelope and thereby influence bacterial cell division. Thus, LTA biosynthesis provides an attractive target for the development of novel antimicrobial interventions. Recent studies, notably two investigations of S. aureus and another of Bacillus subtilis, have greatly improved our understanding of the genetic basis of LTA biosynthesis. In addition, reports have revealed that at least some members of the Actinobacteria (another phylum of Gram-positive bacteria) produce LTAs, rather than the lipoglycans previously assumed to be typical of this taxon. The availability of whole bacterial genome sequences has enabled us to perform comparative analyses to shed light on the distribution of putative LTA biosynthetic genes among bacteria. Here, we discuss the results of these genomic analyses, together with the current literature, and propose that LTA biosynthesis in Actinobacteria might be fundamentally different to that in most Firmicutes.

Structure of the diaminopimelate epimerase DapF from Mycobacterium tuberculosis.

The meso (or D,L) isomer of diaminopimelic acid (DAP), a precursor of L-lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan. While the peptidoglycan incorporated in the highly complex cell wall of the pathogen Mycobacterium tuberculosis structurally resembles that of Escherichia coli, it is unique in that it can contain penicillin-resistant meso-DAP-->meso-DAP linkages. The interconversion of L,L-DAP and meso-DAP is catalysed by the DAP epimerase DapF, a gene product that is essential in M. tuberculosis. Here, the crystal structure of the ligand-free form of M. tuberculosis DapF (MtDapF) refined to a resolution of 2.6 A is reported. MtDapF shows small if distinct deviations in secondary structure from the two-domain alpha/beta-fold of the known structures of Haemophilus influenzae DapF and Bacillus anthracis DapF, which are in line with its low sequence identity (

Sequence and analysis of a plasmid-encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter.

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg(2+) transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.

New drugs and vaccines for drug-resistant Mycobacterium tuberculosis infections.

Tuberculosis remains the most common cause of death due to a single infective organism. Despite the availability of a vaccine and chemotherapeutic options, the global disease burden remains relatively unaffected. The ability of the mycobacterial etiological agents to adopt a semidormant, phenotypically drug-resistant state requires that chemotherapy is both complex and lengthy. The emergence of drug resistance has raised the possibility of virtually untreatable tuberculosis. Furthermore, the currently used bacillus Calmette-Guerin vaccine has had mixed success in protecting susceptible populations. Given this backdrop, the need for novel anti-infectives and more effective vaccines is clearly evident. Recent progress, described herein, has seen the development and entry into clinical trials of several new drugs and vaccine candidates.

Expression, purification and characterisation of soluble GlfT and the identification of a novel galactofuranosyltransferase Rv3782 involved in priming GlfT-mediated galactan polymerisation in Mycobacterium tuberculosis.

The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GlfT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5 mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers [14C]Galf to a range of both beta(1-->5) and beta(1-->6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the beta(1-->6) disaccharide acceptor. However, we could not detect binding or galactofuranosyltransferase activity with an n-octyl beta-d-Gal-(1-->4)-alpha-l-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis.

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization.

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

BACKGROUND: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. METHODOLOGY/PRINCIPLE FINDINGS: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. CONCLUSIONS/SIGNIFICANCE: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

Flavonoid inhibitors as novel antimycobacterial agents targeting Rv0636, a putative dehydratase enzyme involved in Mycobacterium tuberculosis fatty acid synthase II.

Flavonoids comprise a large group of bioactive polyphenolic plant secondary metabolites. Several of these possess potent in vivo activity against Escherichia coli and Plasmodium falciparum, targeting enzymes involved in fatty acid biosynthesis, such as enoyl-ACP-reductase, beta-ketoacyl-ACP reductase and beta-hydroxyacyl-ACP dehydratase. Herein, we report that butein, isoliquirtigenin, 2,2',4'-trihydroxychalcone and fisetin inhibit the growth of Mycobacterium bovis BCG. Furthermore, in vitro inhibition of the mycolic-acid-producing fatty acid synthase II (FAS-II) of Mycobacterium smegmatis suggests a mode of action related to those observed in E. coli and P. falciparum. Through a bioinformatic approach, we have established the product of Rv0636 as a candidate for the unknown mycobacterial dehydratase, and its overexpression in M. bovis BCG conferred resistance to growth inhibition by butein and isoliquirtigenin, and relieved inhibition of fatty acid and mycolic acid biosynthesis in vivo. Furthermore, after overexpression of Rv0636 in M. smegmatis, FAS-II was less sensitive to these inhibitors in vitro. Overall, the data suggest that these flavonoids are inhibitors of mycobacterial FAS-II and in particular Rv0636, which represents a strong candidate for the beta-hydroxyacyl-ACP dehydratase enzyme of M. tuberculosis FAS-II.