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July 7, 2019

Bioinformatics analysis and characterization of highly efficient polyvinyl alcohol (PVA)-degrading enzymes from the novel PVA degrader Stenotrophomonas rhizophila QL-P4.

Polyvinyl alcohol (PVA) is used widely in industry, and associated environmental pollution is a serious problem. Herein, we report a novel, efficient PVA degrader, Stenotrophomonas rhizophila QL-P4, isolated from fallen leaves from virgin forest in the Qinling Mountains. The complete genome was obtained using single-molecule real-time (SMRT) technology and corrected using Illumina sequencing. Bioinformatics analysis revealed eight PVA/OVA (vinyl alcohol oligomer)-degrading genes. Of these, seven genes were predicted to be involved in the classical intracellular PVA/OVA degradation pathway, and one (BAY15_3292) was identified as a novel PVA oxidase. Five PVA/OVA-degrading enzymes were purified and characterised. Among which, BAY15_1712, a PVA dehydrogenase (PVADH), displayed high catalytic efficiency towards PVA and OVA substrate. All reported PVADHs only have PVA-degrading ability. Most importantly, we discovered a novel PVA oxidase (BAY15_3292) that exhibited highest PVA-degrading efficiency than the reported PVADHs. Further investigation indicated that BAY15_3292 plays a crucial role in PVA degradation in S. rhizophila QL-P4. Knocking out BAY15_3292 resulted in a significant decline in PVA-degrading activity in S. rhizophila QL-P4. Interestingly, we found that BAY15_3292 possesses exocrine activity, which distinguishes it from classical PVADHs. Transparent circle experiments further proved that BAY15_3292 greatly affects extracellular PVA degradation in S. rhizophila QL-P4. The exocrine characteristics of BAY15_3292 facilitate its potential application to PVA bioremediation. In addition, we report three new efficient secondary alcohol dehydrogenases (SADHs) with OVA-degrading ability in S. rhizophila QL-P4, compared with only one OVA-degrading SADH as reported previously.Importance With the widespread application of PVA in industry, PVA-related environmental pollution is an increasingly serious issue. Because PVA is difficult to degrade, it accumulates in aquatic environments and causes chronic toxicity to aquatic organisms. Biodegradation of PVA, as an economical and environment-friendly method, has attracted much interest. To date, effective and applicable PVA-degrading bacteria/enzymes have not been reported. Herein, we report a new efficient PVA degrader (S. rhizophila QL-P4) that has five PVA/OVA-degrading enzymes with high catalytic efficiency, among which BAY15_1712 is the only reported PVADH with both PVA- and OVA-degrading abilities. Importantly, we discovered a novel PVA oxidase (BAY15_3292) that is not only more efficient than other reported PVA-degrading PVADHs, but also has exocrine activity. Overall, our findings provide new insight into PVA-degrading pathways in microorganisms, and suggest S. rhizophila QL-P4 and its enzymes have potential for application to PVA bioremediation to reduce or eliminate PVA-related environmental pollution. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Heat resistance mediated by pLM58 plasmid-borne ClpL in Listeria monocytogenes.

Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wild-type L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log10 reduction) than strain AL4E (1.4 CFU/ml log10 reduction) after heating at 55°C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log10 reduction) at 55°C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log10 reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log10 reduction) at 55°C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCEListeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.

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July 7, 2019

Genome-wide discovery of genes required for capsule production by uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant. Copyright © 2017 Goh et al.

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July 7, 2019

Complete genome sequence of Citrobacter werkmanii strain BF-6 isolated from industrial putrefaction.

In our previous study, Citrobacter werkmanii BF-6 was isolated from an industrial spoilage sample and demonstrated an excellent ability to form biofilms, which could be affected by various environmental factors. However, the genome sequence of this organism has not been reported so far.We report the complete genome sequence of C. werkmanii BF-6 together with the description of the genome features and its annotation. The size of the complete chromosome is 4,929,789 bp with an average coverage of 137×. The chromosome exhibits an average G + C content of 52.0%, and encodes 4570 protein coding genes, 84 tRNA genes, 25 rRNA operons, 3 microsatellite sequences and 34 minisatellite sequences. A previously unknown circular plasmid designated as pCW001 was also found with a length of 212,549 bp and a G + C content of 48.2%. 73.5%, 75.6% and 92.6% of the protein coding genes could be assigned to GO Ontology, KEGG Pathway, and COG (Clusters of Orthologous Groups) categories respectively. C. werkmanii BF-6 and C. werkmanii NRBC 105721 exhibited the closest evolutionary relationships based on 16S ribosomal RNA and core-pan genome assay. Furthermore, C. werkmanii BF-6 exhibits typical bacterial biofilm formation and development. In the RT-PCR experiments, we found that a great number of biofilm related genes, such as bsmA, bssR, bssS, hmsP, tabA, csgA, csgB, csgC, csgD, csgE, and csgG, were involved in C. werkmanii BF-6 biofilm formation.This is the first complete genome of C. werkmanii. Our work highlights the potential genetic mechanisms involved in biofilm formation and paves a way for further application of C. werkmanii in biofilms research.

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July 7, 2019

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

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July 7, 2019

Convergence of plasmid architectures drives emergence of multi-drug resistance in a clonally diverse Escherichia coli population from a veterinary clinical care setting.

The purpose of this study was to determine the plasmid architecture and context of resistance genes in multi-drug resistant (MDR) Escherichia coli strains isolated from urinary tract infections in dogs. Illumina and single-molecule real-time (SMRT) sequencing were applied to assemble the complete genomes of E. coli strains associated with clinical urinary tract infections, which were either phenotypically MDR or drug susceptible. This revealed that multiple distinct families of plasmids were associated with building an MDR phenotype. Plasmid-mediated AmpC (CMY-2) beta-lactamase resistance was associated with a clonal group of IncI1 plasmids that has remained stable in isolates collected up to a decade apart. Other plasmids, in particular those with an IncF replicon type, contained other resistance gene markers, so that the emergence of these MDR strains was driven by the accumulation of multiple plasmids, up to 5 replicons in specific cases. This study indicates that vulnerable patients, often with complex clinical histories provide a setting leading to the emergence of MDR E. coli strains in clonally distinct commensal backgrounds. While it is known that horizontally-transferred resistance supplements uropathogenic strains of E. coli such as ST131, our study demonstrates that the selection of an MDR phenotype in commensal E. coli strains can result in opportunistic infections in vulnerable patient populations. These strains provide a reservoir for the onward transfer of resistance alleles into more typically pathogenic strains and provide opportunities for the coalition of resistance and virulence determinants on plasmids as evidenced by the IncF replicons characterised in this study. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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July 7, 2019

Complete genome sequence of indigo-producing bacterium Celeribacter sp. strain TSPH2.

Celeribacter sp. strain TSPH2, a novel producer of indigo, was isolated from oil-contaminated sediment. We present here its genome sequence consisting of one circular chromosome (4 Mb) and one plasmid (0.15 Mb), with an overall G+C content of 60.9%. This strain contains oxygenase genes involved in indigo synthesis, such as flavin-containing monooxygenase. Copyright © 2017 Kim et al.

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July 7, 2019

Complete genomic sequences of two Salmonella enterica subsp. enterica serogroup C2 (O:6,8) strains from Central California.

Salmonella enterica subsp. enterica strains RM11060, serotype 6,8:d:-, and RM11065, serotype 6,8:-:e,n,z15, were isolated from environmental samples collected in central California in 2009. We report the complete genome sequences of these two strains. These genomic sequences are distinct and will provide additional data to our understanding of S. enterica genomics.

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July 7, 2019

An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics.

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations. Our strategy toward accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, ab initio gene prediction algorithms and in silico ORFs (a modified six-frame translation considering alternative start codons) in an integrated proteogenomics database (iPtgxDB) that covers the entire protein-coding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics data set against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and single amino acid variations (SAAVs) identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin. We release iPtgxDBs for B. henselae, Bradyrhizobium diazoefficiens and Escherichia coli and the software to generate both proteogenomics search databases and integrated annotation files that can be viewed in a genome browser for any prokaryote.© 2017 Omasits et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Genomic comparison between Staphylococcus aureus GN strains clinically isolated from a familial infection case: IS1272 transposition through a novel inverted repeat-replacing mechanism.

A bacterial insertion sequence (IS) is a mobile DNA sequence carrying only the transposase gene (tnp) that acts as a mutator to disrupt genes, alter gene expressions, and cause genomic rearrangements. “Canonical” ISs have historically been characterized by their terminal inverted repeats (IRs), which may form a stem-loop structure, and duplications of a short (non-IR) target sequence at both ends, called target site duplications (TSDs). The IS distributions and virulence potentials of Staphylococcus aureus genomes in familial infection cases are unclear. Here, we determined the complete circular genome sequences of familial strains from a Panton-Valentine leukocidin (PVL)-positive ST50/agr4 S. aureus (GN) infection of a 4-year old boy with skin abscesses. The genomes of the patient strain (GN1) and parent strain (GN3) were rich for “canonical” IS1272 with terminal IRs, both having 13 commonly-existing copies (ce-IS1272). Moreover, GN1 had a newly-inserted IS1272 (ni-IS1272) on the PVL-converting prophage, while GN3 had two copies of ni-IS1272 within the DNA helicase gene and near rot. The GN3 genome also had a small deletion. The targets of ni-IS1272 transposition were IR structures, in contrast with previous “canonical” ISs. There were no TSDs. Based on a database search, the targets for ce-IS1272 were IRs or “non-IRs”. IS1272 included a larger structure with tandem duplications of the left (IRL) side sequence; tnp included minor cases of a long fusion form and truncated form. One ce-IS1272 was associated with the segments responsible for immune evasion and drug resistance. Regarding virulence, GN1 expressed cytolytic peptides (phenol-soluble modulin a and d-hemolysin) and PVL more strongly than some other familial strains. These results suggest that IS1272 transposes through an IR-replacing mechanism, with an irreversible process unlike that of “canonical” transpositions, resulting in genomic variations, and that, among the familial strains, the patient strain has strong virulence potential based on community-associated virulence factors.

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July 7, 2019

Complete genome sequencing of Arachidicoccus ginsenosidimutans sp. nov., and its application for production of minor ginsenosides by finding a novel ginsenoside-transforming beta-glucosidase

A novel bacterial strain (BS20T), which has ginsenoside-transforming ability, was whole genome sequenced for the identification of a target gene. After complete genome sequencing, phylogenetic, phenotypic and chemotaxonomic analyses, the strain BS20T (Arachidicoccus ginsenosidimutans sp. nov.) was placed within the genus Arachidicoccus of family Chitinophagaceae. The complete genome of strain BS20T comprised a circular chromosome of 4[thin space (1/6-em)]138[thin space (1/6-em)]017 bp. To find the target functional gene, 17 sets of four different glycoside hydrolases were cloned in E. coli BL21 (DE3) using the pGEX4T-1 vector and were characterized. Among these 17 sets of clones, only one, BglAg-762, exhibited ginsenoside-conversion ability. The BglAg-762 comprised 762 amino acid residues and belonged to the glycoside hydrolase family 3. The recombinant enzyme (GST-BglAg-762) was able to convert major ginsenosides Rb1 to F2 via gypenoside-XVII (Gyp-XVII), Rb2 to C-O, and Rb3, Rc, Rd, and Gyp-XVII to C-Mx1, C-Mc1, and F2, respectively. Finally, ginsenoside F2 was transformed into compound K (C-K). Besides, these pilot data demonstrate the identification of 17 sets of target/functional genes of 4 different glycoside hydrolases from a novel bacterial species via whole genome sequencing. Our results have shown that the recombinant BglAg-762 very quickly converts the major ginsenosides into minor ginsenosides, which can be used for the enhanced production of target minor ginsenosides. Furthermore, the web service of NCBI is suitable for any targeted gene identification, but based on our experimental analysis we concluded that the hypothetical protein present in NCBI should be considered as a putative or uncharacterized protein.

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July 7, 2019

Dissemination and characteristics of a novel plasmid-encoded carbapenem-hydrolyzing class D beta-lactamase, OXA-436 from four patients involving six different hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D carbapenemase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates including Enterobacter asburiae (n=3), Citrobacter freundii (n=2) and Klebsiella pneumoniae (n=1) were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4-92.8% identity to other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to OXA-48 and OXA-181 with activity against penicillins including temocillin, limited or no activity against extended-spectrum cephalosporins and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ~314 kb IncHI2/IncHI2A plasmid belonging to pMLST ST1, in a region surrounded by chromosomal genes previously identified adjacent to blaOXA-genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with similar functional properties as OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and plasmid location of blaOXA-436 illustrates its potential for further dissemination. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Highly accurate fluorogenic DNA sequencing with information theory-based error correction.

Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory-based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.

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July 7, 2019

Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the IncI2, IncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

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July 7, 2019

pSY153-MDR, a p12969-DIM-related mega plasmid carrying blaIMP-45 and armA, from clinical Pseudomonas putida.

This work characterized mega plasmid pSY153-MDR, carrying blaIMP-45 and armA, from a multidrug-resistant (MDR) Pseudomonas putida isolate from the urine of a cerebral infarction patient in China. The backbone of pSY153-MDR was closely related to Pseudomonas plasmids p12969-DIM, pOZ176, pBM413, pTTS12, and pRBL16, and could not be assigned to any of the known incompatibility groups. The accessory modules of pSY153-MDR were composed of 10 individual insertion sequence elements and two different MDR regions, and differed dramatically from the above plasmids. Fifteen non-redundant resistance markers were identified to be involved in resistance to at least eight distinct classes of antibiotics. All of these resistance genes were associated with mobile elements, and were embedded within the two MDR regions. blaIMP-45 and armA coexisted in a Tn1403-Tn1548 region, which was generated from homologous recombination of Tn1403- and Tn1548-like transposons. The second copy of armA was a component of the ISCR28-armA-?ISCR28 structure, representing a novel armA vehicle. This vehicle was located within In48, which was related to In363 and In1058. Data presented here provide a deeper insight into the evolutionary history of SY153, especially in regard to how it became extensively drug-resistant.

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