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Background & objectives Linezolid (LZD) is increasingly being used in tuberculosis (TB) treatment. However, LZD resistance has already been reported, which is highly alarming, given its critical therapeutic role. This study was aimed to phenotypically and genotypically assess LZD resistance in Mycobacterium tuberculosis (MTB) isolates at a laboratory in a tertiary care centre in Mumbai, India. Methods A sample of 32 consecutive LZD-resistant MTB isolates identified by liquid culture susceptibility testing was subjected to whole-genome sequencing (WGS) on the Illumina NextSeq platform. Sequences were analyzed using BioNumerics software to predict resistance for 12 antibiotics within 15 min. Results Sixty eight of the 2179 isolates tested for LZD resistance by MGIT-based susceptibility testing (June 2015 to June 2016) were LZD-resistant. Thirty two consecutive LZD-resistant isolates were analyzed by WGS to screen for known mutations conferring LZD resistance. WGS of 32 phenotypically LZD-resistant isolates showed that C154R in the rplC gene and G2814T in the rrl gene were the major resistance determinants. Interpretation & conclusions LZD resistance poses an important risk to the success of treatment regimens, especially those designed for resistant isolates; such regimens are extensively used in India. As LZD-containing regimens increase in prominence, it is important to support clinical decision-making with an improved understanding of the common mutations conferring LZD resistance and their frequency in different settings.

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealed; however, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen.

Background Bacteria belonging to the genus Haemophilus cause a wide range of diseases in humans. Recently, H. influenzae was classified by the WHO as priority pathogen due to the wide spread of ampicillin resistant strains. However, other Haemophilus spp. are often misclassified as H. influenzae. Therefore, we established an accurate and rapid whole genome sequencing (WGS) based classification and serotyping algorithm and combined it with the detection of resistance genes. Methods A gene presence/absence-based classification algorithm was developed, which employs the open-source gene-detection tool SRST2 and a new classification database comprising 36 genes, including capsule loci for serotyping. These genes were identified using a comparative genome analysis of 215 strains belonging to ten human-related Haemophilus (sub)species (training dataset). The algorithm was evaluated on 1329 public short read datasets (evaluation dataset) and used to reclassify 262 clinical Haemophilus spp. isolates from 250 patients (German cohort). In addition, the presence of antibiotic resistance genes within the German dataset was evaluated with SRST2 and correlated with results of traditional phenotyping assays. Results The newly developed algorithm can differentiate between clinically relevant Haemophilus species including, but not limited to, H. influenzae, H. haemolyticus, and H. parainfluenzae. It can also identify putative haemin-independent H. haemolyticus strains and determine the serotype of typeable Haemophilus strains. The algorithm performed excellently in the evaluation dataset (99.6% concordance with reported species classification and 99.5% with reported serotype) and revealed several misclassifications. Additionally, 83 out of 262 (31.7%) suspected H. influenzae strains from the German cohort were in fact H. haemolyticus strains, some of which associated with mouth abscesses and lower respiratory tract infections. Resistance genes were detected in 16 out of 262 datasets from the German cohort. Prediction of ampicillin resistance, associated with blaTEM-1D, and tetracycline resistance, associated with tetB, correlated well with available phenotypic data. Conclusions Our new classification database and algorithm have the potential to improve diagnosis and surveillance of Haemophilus spp. and can easily be coupled with other public genotyping and antimicrobial resistance databases. Our data also point towards a possible pathogenic role of H. haemolyticus strains, which needs to be further investigated.

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealed; however, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen.

UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource.

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy's from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified enzymes were found to display high-temperature optima (up to 80 °C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 °C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy's which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy's. Finally, the physiological relevance of this enzyme specificity is discussed in the context of metabolic pathways and genomic organization.

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.

Sucrose phosphorylase (SP) is a promising biocatalyst for the production of special sugars and glycoconjugates, but its transglycosylation activity rarely exceeds the competing hydrolytic reaction. Knowing how specificity is controlled, would allow to optimise this activity in an efficient way by means of enzyme engineering. Therefore, in this study, a map of the acceptor site of the SP from Bifidobacterium adolescentis was created by substituting each residue by alanine and analysing the influence on the affinity for both the natural (inorganic phosphate and fructose) and alternative acceptors ( d -arabitol and pyridoxine). All residues examined were found to contribute to the specificity for phosphate (Arg135, Leu343, Tyr344), fructose (Tyr132, Asp342) or both (Pro134, Tyr196, His234, Gln345). Alternative acceptors that are glycosylated rather efficiently (e.g. d -arabitol) were found to interact with the same residues as fructose, whereas poor acceptors like pyridoxine do not seem to make any specific interactions with the enzyme. Furthermore, it is shown here that SP is already optimised to outcompete water as an acceptor substrate, meaning that it will be very difficult to lower its hydrolytic activity any further. Consequently, increasing the transglycosylation activity towards alternative acceptors seems to be the best strategy, although that would probably require a drastic remodelling of the acceptor site in most cases.

Natural product glycosylations by Leloir glycosyltransferases (GTs) require expensive nucleotide-activated sugars as substrates. Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. Coupling of SuSy and GT reactions in one-pot cascade transformations creates a UDP cycle, which regenerates the UDP-glucose continuously and so makes it an expedient donor for glucoside production. Here we compare SuSys with divergent kinetic characteristics for UDP-glucose recycling in the synthesis of the natural C-glucoside nothofagin. Development of a fast reversed-phase ion-pairing HPLC method, quantifying all relevant reactants from the coupled conversion in a single run, was key to dissect the main factors of recycling efficiency. Limitations due to high KM , both for UDP and sucrose, were revealed for the bacterial SuSy from Acidithiobacillus caldus. The L637M-T640V double mutant of this SuSy with a 60-fold reduced KM for UDP substantially improved UDP-glucose recycling. The SuSy from Glycine max (soybean) was nevertheless the most active enzyme at the UDP (≤ 0.5 mM) and sucrose (≤ 1 M) concentrations used. It was also unexpectedly stable at up to 50°C where spontaneous decomposition of UDP-glucose started to become problematic. The herein gained in-depth understanding of requirements for UDP-glucose regeneration supports development of efficient GT-SuSy cascades.

Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower Km value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. All eight single to sextuple mutants had similar activities as the wild-type enzyme but significantly reduced Km values for UDP (up to 60 times). In addition, we recognized that substrate inhibition by UDP is introduced by a methionine at position 637. The affinity for ADP also increased for all but one variant, although the improvement was much smaller compared to UDP. Further characterization of a double mutant also revealed more than 2-fold reduction in Km values for CDP and GDP. This demonstrates the general impact of the motif on nucleotide binding. Furthermore, this research also led to the establishment of a bacterial SuSy variant that is suitable for the recycling of UDP during glycosylation reactions. The latter was successfully demonstrated by combining this variant with a glycosyltransferase in a one-pot reaction for the production of the C-glucoside nothofagin, a health-promoting flavonoid naturally found in rooibos (tea).

Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions.

Recent studies portend a rising global spread and adaptation of human- or healthcare-associated pathogens. Here, we analysed an international collection of the emerging, multidrug-resistant, opportunistic pathogenStenotrophomonas maltophiliafrom 22 countries to infer population structure and clonality at a global level. We show that theS. maltophiliacomplex is divided into 23 monophyletic lineages, most of which harboured strains of all degrees of human virulence. Lineage Sm6 comprised the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identified potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.One Sentence SummaryTheS. maltophiliacomplex comprises genetically diverse, globally distributed lineages with evidence for intra-hospital transmission.