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The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5′-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S -glycosyl- l -cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S -acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure–function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S -acetamide derivative of lincosamide. The reaction mechanisms of the pyridoxal phosphate (PLP)-dependent enzymes LmbF and CcbF—involved in the biosynthesis of the antibiotic lincosamide—are characterized here. Structure–function analyses reveal structural features responsible for S -alkyl moiety diversification in lincosamides. Using structure- and calculation-based enzyme engineering, the selectivity profiles of these enzymes are designed to generate an unnatural lincosamide derivative.

This study was conducted to estimate the prevalence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) in retail chicken meat and broiler chickens from the Province of Quebec, Canada, and to characterize LA-MRSA isolates. A total of 309 chicken drumsticks and thighs were randomly selected in 2013 from 43 retail stores in the Monteregie. In addition, nasal swabs and caeca samples were collected in 2013-2014 from 200 broiler chickens of 38 different flocks. LA-MRSA was not detected in broiler chickens. Fifteen LA-MRSA isolates were recovered from four (1.3%) of the 309 chicken meat samples. Multi-Locus Sequence Typing (MLST) and SCCmec typing revealed two profiles (ST398-MRSA-V and ST8-MRSA-IVa), which were distinct using pulse-field gel electrophoresis (PFGE) and microarray (antimicrobial resistance and virulence genes) analyses. In addition to beta-lactam resistance, tetracycline and spectinomycin resistance was detected in all isolates from the 3 positive samples of the ST398 profile. Southern blot hybridization revealed that the resistance genes aad(D) and lnu(A), encoding resistances to aminoglycosides and lincosamides respectively, were located on plasmid. All isolates were able to produce biofilms, but biofilm production was not correlated with hld gene expression. Our results show the presence of two separate lineages of MRSA in retail chicken meat in Quebec, one of which is likely of human origin.

The structure of lincomycin A consists of the unusual eight-carbon thiosugar core methyllincosamide (MTL) decorated with a pendent N-methylprolinyl moiety. Previous studies on MTL biosynthesis have suggested GDP-D-erythro-α-D-gluco-octose and GDP-D-α-D-lincosamide as key intermediates in the pathway. However, the enzyme-catalyzed reactions resulting in the conversion of GDP-D-erythro-α-D-gluco-octose to GDP-D-α-D-lincosamide have not yet been elucidated. Herein, a biosynthetic subpathway involving the activities of four enzymes—LmbM, LmbL, CcbZ, and CcbS (the LmbZ and LmbS equivalents in the closely related celesticetin pathway)—is reported. These enzymes catalyze the previously unknown biosynthetic steps including 6-epimerization, 6,8-dehydration, 4-epimerization, and 6-transamination that convert GDP-D-erythro-α-D-gluco-octose to GDP-D-α-D-lincosamide. Identification of these reactions completes the description of the entire lincomycin biosynthetic pathway. This work is significant since it not only resolves the missing link in octose core assembly of a thiosugar-containing natural product but also showcases the sophistication in catalytic logic of enzymes involved in carbohydrate transformations.

Existing recommended first-line antibiotic agents for MRSA pneumonia have several shortcomings. We reviewed 29 cases of community- and hospital-acquired MRSA pneumonia managed at our hospital. Lincosamide monotherapy was administered to 21/29 (72%) and was the predominant antibiotic regimen (> 50% course duration) in 19/29 (66%). Patients receiving lincosamide-predominant monotherapy were no more likely to die or require intensive care unit admission than patients receiving vancomycin-predominant monotherapy (5/19 (26%) versus 4/7 (57%), p = 0.19); 5/7 (71%) patients admitted to ICU and 4/5 (80%) bacteraemic patients received lincosamide-predominant monotherapy. MRSA pneumonia can be safely treated with lincosamide monotherapy if the isolate is susceptible.

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of the hflX gene in the pathogenic Mycobacterium abscessus, as well as the nonpathogenic Mycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided by Mab_hflX is equivalent to that conferred by erm41, implying that hflX constitutes a significant resistance determinant in M. abscessus. We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in a ΔMs_hflX strain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence of hflX genes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.

Background The microbiome of the oral cavity is the second-largest and diverse microbiota after the gut, harboring over 700 species of bacteria and including also fungi, viruses, and protozoa. With its diverse niches, the oral cavity is a very complex environment, where different microbes preferentially colonize different habitats. Recent data indicate that the oral microbiome has essential functions in maintaining oral and systemic health, and the emergence of 16S rRNA gene next-generation sequencing (NGS) has greatly contributed to revealing the complexity of its bacterial component. However, a detailed site-specific map of oral microorganisms (including also eukaryotes and viruses) and their relative abundance is still missing. Here, we aimed to obtain a comprehensive view of the healthy oral microbiome (HOM), including its drug-resistance features. Results The oral microbiome of twenty healthy subjects was analyzed by whole-genome sequencing (WGS) and real-time quantitative PCR microarray. Sampled oral micro-habitat included tongue dorsum, hard palate, buccal mucosa, keratinized gingiva, supragingival and subgingival plaque, and saliva with or without rinsing. Each sampled oral niche evidenced a different microbial community, including bacteria, fungi, and viruses. Alpha-diversity evidenced significant differences among the different sampled sites ( p  < 0.0001) but not among the enrolled subjects ( p  = 0.876), strengthening the notion of a recognizable HOM. Of note, oral rinse microbiome was more representative of the whole site-specific microbiomes, compared with that of saliva. Interestingly, HOM resistome included highly prevalent genes conferring resistance to macrolide, lincosamides, streptogramin, and tetracycline. Conclusions The data obtained in 20 subjects by WGS and microarray analysis provide for the first time a comprehensive view of HOM and its resistome, contributing to a deeper understanding of the composition of oral microbiome in the healthy subject, and providing an important reference for future studies, allowing to identify microbial signatures related to functional and metabolic alterations associated with diseases, potentially useful for targeted therapies and precision medicine.

Malaria, a parasite vector-borne disease, is one of the biggest health threats in tropical regions, despite the availability of malaria chemoprophylaxis. The emergence and rapid extension of Plasmodium falciparum resistance to various anti-malarial drugs has gradually limited the potential malaria therapeutics available to clinicians. In this context, macrolides and associated antibiotics based on similar mechanism of action like lincosamides constitute an interesting alternative in the treatment of malaria. These molecules, whose action spectrum is similar to that of tetracyclines, are typically administered to children and pregnant women. Recent studies have examined the effects of azithromycin and the lincosamide clindamycin, on isolates from different continents. Azithromycin and clindamycin are effective and well tolerated in the treatment of uncomplicated malaria in combination with quinine. This literature review assesses the roles of macrolides and lincosamides in the prophylaxis and treatment of malaria.

Knowing about the antibiotic resistance, serotypes, and virulence-associated genes of Group B Streptococcus for epidemiological and vaccine development is very important. We have determined antimicrobial susceptibility patterns, serotype, and virulence profiles. The antibiotic susceptibility was assessed for a total of 421 Streptococcus agalactiae strains, isolated from pregnant women and neonates. Then, 89 erythromycin and/or clindamycin-resistant strains (82 isolates obtained from pregnant women and seven isolates derived from neonates) were assessed in detail. PCR techniques were used to identify the studied strains, perform serotyping, and assess genes encoding selected virulence factors. Phenotypic and genotypic methods determined the mechanisms of resistance. All tested strains were sensitive to penicillin and levofloxacin. The constitutive MLS B mechanism (78.2%), inducible MLS B mechanism (14.9%), and M phenotype (6.9%) were identified in the macrolide-resistant strains. It was found that macrolide resistance is strongly associated with the presence of the ermB gene and serotype V. FbsA, fbsB, fbsC, scpB , and lmb formed the most recurring pattern of genes among the nine surface proteins whose genes were analysed. A minority (7.9%) of the GBS isolates exhibited resistance to lincosamides and macrolides, or either, including those that comprised the hypervirulent clone ST-17. The representative antibiotic resistance pattern consisted of erythromycin, clindamycin, and tetracycline resistance (71.9%). An increase in the fraction of strains resistant to macrolides and lincosamides indicates the need for monitoring both the susceptibility of these strains and the presence of the ST-17 clone.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern 1 . For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings 2 . Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the m 2 6 A 2058 nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance. Structure-guided design and component-based synthesis are used to produce iboxamycin, a novel ribosome-binding antibiotic with potent activity against Gram-positive and Gram-negative bacteria.

Background Gram-positive Streptococci is a huge group of different species that are classified based on its hemolytic effect besides the C-substance in the cell wall. This study focuses on the investigation of the prevalence and genetic basis of resistance to macrolides, lincosamides, and streptogramins (MLS) in α- and β-hemolytic Streptococci . Methods Streptococcal isolates were identified and their resistance was assessed to MLS antibiotics through phenotypic analysis and genotypic screening of resistance genes. Isolates were also tested for susceptibility to antiseptics/disinfectants. The correlation between high MLS antibiotic resistance and reduced susceptibility to biocides was assessed. Efflux pump activity in the most resistant isolates (to both MLS antibiotics and biocides) was investigated. Results The susceptibility testing indicates an increasing resistance to MLS, particularly macrolides (erythromycin, azithromycin, and clarithromycin) and lincomycin. By screening the resistance, the most predominant phenotype is the constitutive (cMLS) one, while the erm genes, particularly ermB , are the most detected genotype. Furthermore, the esterase-encoding gene ereA is widely distributed in the streptococcal isolates. By evaluating the minimum inhibitory concentrations (MICs) to different biocides, there was a strong relation between the increased MIC values to both MLS antibiotics and tested biocides. This can be attributed mainly to the transferable ermB gene and the enhanced bacterial efflux. Conclusions A significant correlation exists between reduced biocide susceptibility and resistance to MLS antibiotics. Elevated efflux pump activity in MLS-resistant isolates suggests efflux mechanisms may contribute to dual resistance to antibiotics and biocides. However, cross-resistance is primarily driven by the horizontally transferable ermB gene, which confers resistance by targeting the 50S ribosomal subunit.