Rapid Annotation Using Subsystem Technology Research Articles

High-quality genome sequence assembly of R.A73 Enterococcus faecium isolated from freshwater fish mucus

BackgroundWhole-genome sequencing using high throughput technologies has revolutionized and speeded up the scientific investigation of bacterial genetics, biochemistry, and molecular biology. Lactic acid bacteria (LABs) have been extensively used in fermentation and more recently as probiotics in food products that promote health. Genome sequencing and functional genomics investigations of LABs varieties provide rapid and important information about their diversity and their evolution, revealing a significant molecular basis.This study investigated the whole genome sequences of the Enterococcus faecium strain (HG937697), isolated from the mucus of freshwater fish in Tunisian dams. Genomic DNA was extracted using the Quick-GDNA kit and sequenced using the Illumina HiSeq2500 system. Sequences quality assessment was performed using FastQC software. The complete genome annotation was carried out with the Rapid Annotation using Subsystem Technology (RAST) web server then NCBI PGAAP.ResultsThe Enterococcus faecium R.A73 assembled in 28 contigs consisting of 2,935,283 bps. The genome annotation revealed 2884 genes in total including 2834 coding sequences and 50 RNAs containing 3 rRNAs (one rRNA 16 s, one rRNA 23 s and one rRNA 5 s) and 47 tRNAs. Twenty-two genes implicated in bacteriocin production are identified within the Enterococcus faecium R.A73 strain.ConclusionData obtained provide insights to further investigate the effective strategy for testing this Enterococcus faecium R.A73 strain in the industrial manufacturing process. Studying their metabolism with bioinformatics tools represents the future challenge and contribution to improving the utilization of the multi-purpose bacteria in food.

Draft genome and secondary metabolite biosynthetic gene clusters of Streptomyces sp. strain 196

Emergence of MDR 'superbugs' inflamed a severe sense of urgency amongst scientists aiming at the discovery of novel potential drug molecules. Bacteria of the genus Streptomyces are really worth investigating for their immense potential to produce natural compounds of pharmaceutical importance. In the present study, the genome of Streptomyces sp. strain 196 was sequenced, studied and secondary metabolite biosynthetic gene clusters (smBGCs) were detected. FAME analysis was used for taxonomic validation of strain 196. Genome of strain 196 was sequenced using the Illumina NextSeq system which has resulted in a draft genome of 7.4Mb. Rapid annotation using subsystem technology (RAST) results revealed the presence of 6682 CDS, 64 tRNA genes and 7 rRNA genes. Comparative studies revealed that strain 196 have 93.5% nucleotide and 96% protein level similarities with Streptomyces rhizosphaericola 1AS2c. Genome mining using antiSMASH predicted the presence of BGCs responsible for diverse bioactive compound production. The detected gene clusters were two PKS-III, one PKS-I, five NRPS, two hybrid PKS-I/NRPS, one thiopeptide/LAP, and one bacteriocin types. Furthermore, many other types BGCs such as three ectoine, two siderophore, one arylpolyene, two butyrolactone, one lassopeptide, one lanthipeptide and one melanin were also found. The results of this study provides information about genome and BGCs of strain 196, this information is valuable for researchers who are interested in isolation of bioactive compounds and working on heterologous expression of cryptic BGCs for novel bioactive compounds production.

Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242).

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

Integrated genome-based probiotic relevance and safety evaluation of Lactobacillus reuteri PNW1.

This study evaluates whole-genome sequence of Lactobacillus reuteri PNW1 and identifies its safety genes that may qualify it as a putative probiotic. It further extracted the bacteriocin produced by the strain and tested its effectiveness against pathogenic STEC E. coli O177. The genomic DNA was sequenced on illuminal Miseq instrument and the sequenced data was assessed for quality reads before assembled with SPAdes. The draft assembly was annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Further downstream analyses were carried out using appropriate bioinformatic tools. Production of biogenic amines was biochemically confirmed through HPLC analysis. The assembled genome was 2,430,215 bp long in 420 contigs with 39% G+C content. Among all known genes, putatively responsible for the production of toxic biochemicals, only arginine deiminase (EC3.5.3.6) was spotted. Coding sequences (CDS) putative for D-lactate dehydrogenase (EC1.1.1.28), L-lactate dehydrogenase (EC1.1.1.27) and bacteriocin helveticin J were found within the genome together with plethora of other probiotic important genes. The strain harbours only resistant genes putative for Lincosamide (lnuC) and Tetracycline resistant genes (tetW). There was no hit found for virulence factors and probability of the strain being a human pathogen was zero. Two intact prophage regions were detected within the genome of L. reuteri PNW1 and nine CDS were identified for insertion sequence by OASIS which are belong to seven different families. Five putative CDS were identified for the CRISPR, each associated with Cas genes. Maximum zone of inhibition exhibited by the bacteriocin produced L. reuteri PNW1 is 20.0±1.00 mm (crude) and 23.3±1.15 mm (at 0.25 mg/ml) after being partially purified. With the strain predicted as non-human pathogen, coupled with many other identified desired features, L. reuteri PNW1 stands a chance of making good and safe candidates for probiotic, though further in-vivo investigations are still necessary.

Whole genome sequence of an Escherichia coli ST131 strain isolated from a patient with bloodstream infection in China co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6’)-Ib-cr genes

ObjectivesEscherichia coli sequence type 131 (ST131) is an international multiresistant high-risk clone associated with a large number of clinical infections. Here we report the draft genome sequence of a ST131 clinical isolate (EC538) obtained from a patient with bloodstream infection (BSI) in China co-harbouring the blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6ʹ)-Ib-cr genes. MethodsAntimicrobial susceptibility testing of E. coli EC538 was performed. DNA of E. coli EC538 was extracted and was sequenced using an Illumina HiSeqTM X Ten platform. Generated sequence reads were assembled using CLC Genomics Workbench. Contigs were annotated using Rapid Annotation using Subsystem Technology (RAST), and further bioinformatics analyses were performed. ResultsThe total number of assembled bases was 5 420 040bp, with 5611 protein-coding sequences. Escherichia coli EC538 belongs to ST131 by multilocus sequence typing (MLST). The presence of blaKPC-2, blaCTX-M-3 and blaCTX-M-14 genes was detected in addition to other antimicrobial resistance genes conferring resistance to fluoroquinolones, aminoglycosides, trimethoprim, sulfonamides, tetracyclines, macrolides and rifampicin. ConclusionTo our knowledge, this is the first report of an E. coli ST131 strain from a BSI co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6ʹ)-Ib-cr genes in China. The presented genome sequence of a carbapenemase-producing E. coli ST131 strain could provide further insight into the genomic diversity of this highly virulent, multiresistant and successfully pandemic bacterial pathogen.

Outlook of fosmid library from metagenomic of the symbionts associated with coral Siderastrea stellata: structural and functional screening for metabolic and antimicrobial activity

Microorganisms colonize the corals and produce compounds with interesting biological properties for biotechnology. The aim of the present study was to search for biocompounds with industrial potential through the cloning metagenomic DNA of symbionts the coral S. stellata in fosmid vector. The metagenomic library was analyzed using functional screening and sequencing via the Illumina MiSeq. Of the 3648 clones, eight were identified as proteolytic and six as amylolytic. The proteolytic clone, P07H3, also exhibited antimicrobial activity against S. aureus, S Enterica PT11, S Enterica PT4 and S Typhi. No clones were positive for lipase/esterase. Beta-lactamase was a single subsystem identified by Rapid Annotation using Subsystem Technology (RAST) through fosmid sequencing of clone P07H3. However, there were identifications of enzymes that participate in metabolic processes such as polysaccharide catabolism, oxidation-reduction, signal transduction and phosphorylation. When the hypothetical proteins were re-analyzed they exhibited a defined functional domain. All the identified open reading frames (ORFs) had low identity with proteins deposited in BLASTp. The sequencing shows that most of the genomic fragments of clone P07H3 possibly consist of new proteins. The Proteobacteria phylum had the greatest predominance among the analyzed fragments. This is the first report on the prospection of amylases, proteases and antimicrobials of coral S. stellata samples. The analyzes of this study help to improve the knowledge about the metabolic diversity of S. stellata still little explored and viable in the future study of the identified compounds.

Whole genome sequence of an Escherichia coli ST410 isolate co-harbouring blaNDM-5, blaOXA-1, blaCTX-M-15, blaCMY-2, aac(3)-IIa and aac(6')-Ib-cr genes isolated from a patient with bloodstream infection in China.

Mortality associated with carbapenemase-producing Enterobacteriaceae is high as there are few therapeutic options. Escherichia coli sequence type 410 (ST410) is currently an international high-risk clone and is responsible for a large number of clinical infections. Here we report the draft genome sequence of a ST410 clinical E. coli isolate (ECS9) co-harbouring blaNDM-5, blaOXA-1, blaCTX-M-15, blaCMY-2, aac(3)-IIa and aac(6')-Ib-cr genes, obtained from a patient with bloodstream infection in China. The genome of E. coli ECS9 was sequenced using an Illumina HiSeqTM 4000 instrument with a 150-bp paired-end approach. Generated sequence reads were assembled using Velvet 1.2.10. Contigs were annotated using Rapid Annotation using Subsystem Technology (RAST), and further whole-genome sequence data analyses were performed. Escherichia coli ECS9 belongs to multilocus sequence typing (MLST) ST410. The total number of assembled bases was 4 935 145 bp, with 5077 protein-coding sequences. The presence of the blaNDM-5, blaOXA-1, blaCTX-M-15 and blaCMY-2 genes was detected in addition to other antimicrobial resistance genes conferring resistance to fluoroquinolones, aminoglycosides, trimethoprim, sulfonamides and tetracyclines. To our knowledge, this is the first report of anE. coli ST410 strain co-harbouring blaNDM-5, blaOXA-1, blaCTX-M-15, blaCMY-2, aac(3)-IIa and aac(6')-Ib-cr, obtained from a bloodstream infection in China. The presented genome sequence of carbapenemase-producing E. coli strain ST410 could provide further insight into the acquisition of multiple resistance genes by this successful lineage.

Emergence of Klebsiella pneumoniae ST11 co-producing NDM-1 and OXA-48 carbapenemases in Greece.

Klebsiella pneumoniae is a well-known pathogen frequently implicated in serious life-threatening nosocomial infections. Here we present a K. pneumoniae isolate (AHEPA1046) co-harbouring blaNDM-1 and blaOXA-48 isolated from a blood sample of an inpatient in Thessaloniki, Greece. Whole-genome sequencing (WGS) was performed using an Illumina MiniSeq Sequencing System. Multilocus sequence typing (MLST) was performed using a BLAST-based approach, and antimicrobial resistance genes and plasmid replicons were identified by ResFinder and PlasmidFinder, respectively. The Rapid Annotation using Subsystem Technology (RAST) v.2.0 server was used for genome annotation. WGS analysis revealed the complete resistome of K. pneumoniae AHEPA1046. The strain harboured blaNDM-1 and blaOXA-48 together with 16 additional antimicrobial resistance genes and was resistant to carbapenems, aminoglycosides, quinolones, macrolides, tetracyclines, trimethoprim, fosfomycin and phenicols. Moreover, it was classified as ST11. This is the first report of a K. pneumoniae clinical isolate from Greece co-producing NDM-1 and OXA-48 carbapenemases and is one of a few reported worldwide.

Genomic characterisation of a colistin-resistant Klebsiella pneumoniae ST11 strain co-producing KPC-2, FloR, CTX-M-55, SHV-12, FosA and RmtB causing a lethal infection.

The aim of this study was to characterise the genomic and phenotypic characteristics of a colistin-resistant Klebsiella pneumoniae isolate causing a lethal infection that was phenotypically resistant to carbapenems and colistin. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform. Genome annotation was performed by the Rapid Annotation using Subsystem Technology (RAST) server. Antimicrobial resistance genes and plasmid replicons were identified using ResFinder 2.1 and PlasmidFinder 1.3, respectively. The isolate was further characterised by plasmid analysis using S1-PFGE and Southern blot hybridisation with a digoxigenin-labelled probe specific for blaKPC. RESULTS: The genome of K. pneumoniae LSK16 consists of a 6.02-Mbp chromosome and one plasmid. Multilocus sequence typing (MLST) identified the isolate as ST11, a close variant of the international pandemic clone ST258. The isolate was found to harbour blaKPC-2, blaSHV-12, blaCTX-M-55, floR and rmtB genes. Of note, a novel fosfomycin resistance glutathione transferase variant was confirmed by PCR and sequencing, with 98.6% (136/138) identity to fosA. Moreover, amino acid substitutions in PmrB (R256G) and PhoQ (D150G) were identified in LSK16, confirming the polymyxin/colistin resistance, although the isolate was negative for mcr genes. Southern blotting and plasmid analysis revealed that the blaKPC-2 gene was harboured on a non-conjugative IncR plasmid (˜165kb). Here we identified a colistin-resistant K. pneumoniae ST11 strain co-producing KPC-2, FloR, CTX-M-55, SHV-12 and RmtB causing a lethal infection. This study provides new genomic insights into the diversity of K. pneumoniae ST11 prevalent in Zhejiang Province, China.

Whole-genome sequencing study of KPC-encoding Klebsiella pneumoniae isolated in Greek private laboratories from non-hospitalised patients

Greece is endemic for KPC-encoding Klebsiella pneumoniae; however, until now, reports have referred only to hospital isolates. In this study, seven KPC-encoding K. pneumoniae isolated in private laboratories from non-hospitalised patients were characterised. Whole-genome sequencing was performed on an Illumina MiniSeq Sequencing System. Multilocus sequence typing (MLST) was performed using a BLAST-based approach, and antimicrobial resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder, respectively. The Rapid Annotation using Subsystem Technology (RAST) v.2.0 server was used for genome annotation of virulence, pathogenesis and defence genes. Six isolates belonged to the major MLST sequence type 258 (ST258) and one to ST39. The resistome included genes encoding resistance mechanisms to β-lactams, aminoglycosides, quinolones, sulfonamides, trimethoprim, fosfomycin and phenicols, conferring multidrug-resistant phenotypes. Moreover, various genes involved in virulence, pathogenesis and defence have been identified. It is highly probable that these isolates were acquired during previous hospitalisation in Greek hospitals. The presence of KPC-encoding K. pneumoniae in non-hospitalised patients is alarming, although it is not yet possible to assess its actual impact.

Complete genome sequence of an IMP-8, CTX-M-14, CTX-M-3 and QnrS1 co-producing Enterobacter asburiae isolate from a patient with wound infection.

The aim of this study was to investigate the characteristics and complete genome sequence of an IMP-8, CTX-M-14, CTX-M-3 and QnrS1 co-producing multidrug-resistant Enterobacter asburiae isolate (EN3600) from a patient with wound infection. Species identification was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase genes were identified by PCR and Sanger sequencing. The complete genome sequence of E. asburiae EN3600 was obtained using a PacBio RS II platform. Genome annotation was done by Rapid Annotation using Subsystem Technology (RAST) server. Acquired antimicrobial resistance genes (ARGs) and plasmid replicons were detected using ResFinder 2.1 and PlasmidFinder 1.3, respectively. The genome of E. asburiae EN3600 consists of a 4.8-Mbp chromosome and five plasmids. The annotated genome contains various ARGs conferring resistance to aminoglycosides, β-lactams, fluoroquinolones, fosfomycin, macrolides, phenicols, rifampicin and sulfonamides. In addition, plasmids of incompatibility (Inc) groups IncHI2A, IncFIB(pECLA), IncFIB(pQil) and IncP1 were identified. The genes blaIMP-8, blaCTX-M-14 and blaCTX-M-3 were located on different plasmids. The blaIMP-8 gene was carried by an 86-kb IncFIB(pQil) plasmid. The blaCTX-M-3 and qnrS1 genes were co-harboured by an IncP1 plasmid. In addition, blaCTX-M-14 was associated with blaTEM-1B, blaOXA-1, catB3 and sul1 genes in a 116-kb non-typeable plasmid. To our knowledge, this is the first complete genome sequence of an E. asburiae isolate co-producing IMP-8, CTX-M-14, CTX-M-3 and QnrS1. This genome may facilitate the understanding of the resistome, pathogenesis and genomic features of Enterobacter cloacae complex (ECC) and will provide valuable information for accurate identification of ECC.

Genomic analysis of Indian strains of Salmonella enterica subsp. enterica serovar Typhi indicates novel genetic repertoire for pathogenicity and adaptations.

In the era of emerging antibiotic resistance, Salmonella enterica subsp. enterica serovar Typhi the causative agent of typhoid, is a threat for healthcare systems in developing countries especially India, where the disease is highly endemic. Genetic diversity among different strains may be the cause of variable severity of disease in different regions of the world. To explore this genetic diversity, genome annotation by rapid annotation using subsystem technology (RAST) was carried out for genomes of four Salmonella Typhi strains from two distinct areas available in thepublic domain. Two clinical strains were from India (P-stx-12 and E02-1180) and the other two strains considered as reference strains were from the endemic regions of Papua New Guinea (UJ308A and UJ816A). We report that Indian clinical strains possess several similar genes responsible for virulence and pathogenicity as those present in the reference strains. Interestingly, Indian clinical strains also possess 34 additional potential virulence genes that are absent in thereference strains, suggesting the more dreadful nature of Indian clinical strains as compared to those causing endemic typhoid. Indian strains contained genes coding for; Colicin V and bacteriocin production; multidrug resistance efflux pumps; ABC transporters; Type III and Type VI secretion systems, siderophore aerobactin, pathogenicity islands and Vi polysaccharide biosynthesis and transport. These unique genes are also reported in the genomes of other six clinical strains of India analyzed through RAST and IslandViewer 4 for validation purpose. This study highlights the presence of potential genes as molecular targets to overcome the future endemic outbreaks in India.

Nanopore ultra-long read sequencing technology for antimicrobial resistance detection in Mannheimia haemolytica

Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were sequenced at 146× and 111× coverage, respectively with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including β-lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Resistance phenotypes of the M. haemolytica strains were determined by minimum inhibitory concentration (MIC) of the antibiotics. Sequencing with a highly portable MinION device corresponded to MIC assays with most of the antimicrobial resistant determinants being identified with as few as 5437 reads, except for the genes responsible for resistance to Fluoroquinolones. The resulting quality assemblies and AMR gene annotation highlight the efficiency of ultra-long read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

Draft Genome Sequence of Ciprofloxacin and Ceftriaxone Resistant Campylobacter jejuni MM26-781 Assigned to Novel ST Isolated From Common Pigeon in Lithuania.

Campylobacter jejuni is an important zoonotic pathogen known to be resistant to a wide range of antibiotics worldwide. Campylobacter jejuni may be intrinsically resistant to antibiotics or can acquire antibiotic resistance determinants through gene transfer. However, the knowledge of molecular mechanisms of antimicrobial resistance among Campylobacter isolates from wild birds, especially in Lithuania, is limited. Whole genome sequencing (WGS) is a tool for better understanding the evolutionary and epidemiologic dynamics of C jejuni. This study describes a draft whole genome sequence of C jejuni MM26-781 isolated from a common pigeon (Columba livia) in Lithuania in 2011 and assigned to ST-6424 (CC179) sequence type. The draft genome sequence contained 1.68 Mb, comprising 1651 coding genes, 40 transfer RNAs, 1 ribosomal RNA, and 69 pseudogenes with an average G + C content of 30.4%. The RAST (Rapid Annotation using Subsystem Technology) pipeline annotated (NCTC11168) a total of 305 subsystems in the genome of C jejuni MM26-781 strain, with most of the genes associated with amino acids and derivatives related to metabolism (18.93%) and protein metabolism (14.43%). The genes and mutations related to antibiotic resistance, including gyrA and gyrB genes associated with quinolone resistance, blaOXA-448 gene (locus tag C9371_07715) associated with resistance to β-lactams, rpoB gene associated with resistance to rifamycin, vgaE gene associated with resistance to streptogramin and efflux system CmeABC (cmeA, cmeB, cmeC), efflux pump PmrA, and transcriptional regulator CmeR responsible for multidrug resistance in C jejuni MM26-781 chromosome, were identified. Also, the virulence factors, including ciaB, cadF, ceuE, pldA, motB, and bd1A genes, were identified by WGS data analysis.

Draft genome sequence of Xenophilus sp. E41 isolated from an activated sludge reactor treating wastewater with high cephalexin concentration.

This study reports the draft genome sequence of Xenophilus sp. E41, a strain resistant to multiple antimicrobials isolated from an activated sludge reactor treating wastewater with a high cephalexin concentration. Genomic DNA of Xenophilus sp. E41 was extracted and sequenced using an Illumina NovaSeq 6000 system. The generated sequence reads were assembled using MEGAHIT in combination with SOAPdenovo. Mauve and CompareM were used to align the Xenophilus sp. E41 genome to other draft genomes of the genus Xenophilus in order to determine their evolutionary relationships. The draft genome was annotated using the Rapid Annotation using Subsystem Technology (RAST) server and the nr database, whilst antimicrobial resistance genes (ARGs) were identified using the SARG 2.0 database, RAST server and nr database. Xenophilus sp. E41, with a genome length of 5919552bp, harbours seven types of ARGs involving resistance to β-lactams, tetracycline, aminoglycosides, sulfonamides, chloramphenicol, teicoplanin and bleomycin. No virulence factors or plasmids were identified. The genome sequence reported here will provide useful information for a better understanding of antimicrobial resistance profiles in this strain and the genus Xenophilus.

Putative Iron Acquisition Systems in Stenotrophomonas maltophilia.

Iron has been shown to regulate biofilm formation, oxidative stress response and several pathogenic mechanisms in Stenotrophomonas maltophilia. Thus, the present study is aimed at identifying various iron acquisition systems and iron sources utilized during iron starvation in S. maltophilia. The annotations of the complete genome of strains K279a, R551-3, D457 and JV3 through Rapid Annotations using Subsystems Technology (RAST) revealed two putative subsystems to be involved in iron acquisition: the iron siderophore sensor and receptor system and the heme, hemin uptake and utilization systems/hemin transport system. Screening for these acquisition systems in S. maltophilia showed the presence of all tested functional genes in clinical isolates, but only a few in environmental isolates. NanoString nCounter Elements technology, applied to determine the expression pattern of the genes under iron-depleted condition, showed significant expression for FeSR (6.15-fold), HmuT (12.21-fold), Hup (5.46-fold), ETFb (2.28-fold), TonB (2.03-fold) and Fur (3.30-fold). The isolates, when further screened for the production and chemical nature of siderophores using CAS agar diffusion (CASAD) and Arnows’s colorimetric assay, revealed S. maltophilia to produce catechol-type siderophore. Siderophore production was also tested through liquid CAS assay and was found to be greater in the clinical isolate (30.8%) compared to environmental isolates (4%). Both clinical and environmental isolates utilized hemoglobin, hemin, transferrin and lactoferrin as iron sources. All data put together indicates that S. maltophilia utilizes siderophore-mediated and heme-mediated systems for iron acquisition during iron starvation. These data need to be further confirmed through several knockout studies.

Whole-genome sequencing of a CTX-M-11-encoding and quinolone-non-susceptible Klebsiella pneumoniae ST194 isolate from a hospitalised dog in Greece.

The emergence and spread of transferable β-lactamases among Enterobacteriaceae is a major problem both to human and veterinary medicine and is an important contributing factor to the development of multidrug-resistant bacterial isolates. In the present study, whole-genome sequencing of a Klebsiella pneumoniae isolate (LKP817909) resistant to first- and second-generation cephalosporins and non-susceptible to fluoroquinolones, isolated from a urine sample of a hospitalised dog, was performed. Genome sequencing was performed on an Illumina MiniSeq Sequencing System. Multilocus sequence typing (MLST) was performed using a BLAST-based approach, whereas antimicrobial resistance genes and plasmid replicons were identified by ResFinder and PlasmidFinder, respectively. The Rapid Annotation using Subsystem Technology (RAST) server v.2.0 was used for genome annotation. Data analyses revealed the complete resistome of isolate LKP817909, which included the cefotaximase-München-11 (CTX-M-11) extended-spectrum β-lactamase together with 11 other resistance genes. Ten resistance genes were located on plasmids and two on the chromosome. To the best of our knowledge, this is the first detection of a CTX-M-11-producing K. pneumoniae isolated from a canine. The whole genome sequence of the isolate has been deposited at GenBank to serve as a future reference.

Genome sequence and analysis of Mycobacterium tuberculosis strain SWLPK.

Multidrug-resistant Mycobacterium tuberculosis poses a global threat, particularly in developing countries such as Pakistan. Genome sequencing, comparative genomic analysis and drug resistance gene analysis could be beneficial for understanding and monitoring M. tuberculosis disease severity in Pakistan by elucidating the biology of M. tuberculosis. Here the draft genome of M. tuberculosis strain SWLPK was sequenced, assembled and annotated using an Illumina MiSeq system. De novo genomic assembly was conducted using Geneious Pro™ v.10. The assembled genome of strain SWLPK was annotated using the Rapid Annotation using Subsystem Technology (RAST) server, tRNAscan-SE 1.21 and RNAmmer v.1.2, which provide high-quality functional annotation. Mycobacterium tuberculosis strain SWLPK yielded an average read depth of 68.5-fold, which covered 97% of the genome of reference strain H37Rv. The genome contains 4305 protein-coding genes, including key drug resistance and virulence-associated genes such as type seven secretion systems. Additionally, it has a 65.6% GC content and contains 48 RNAs and 12 contigs. We determined that all proteins encoded by this strain contain conserved domains, except OxyR, which is associated with first-line antituberculosis drugs such as ethambutol, rifampicin, streptomycin, pyrazinamide and isoniazid. This genome sequence provides information regarding the drug resistance genes and virulence propensity of M. tuberculosis strain SWLPK. Strain SWLPK appears to be multidrug-resistant, similar to the Beijing genotype, as it clusters in the same group. These findings will pave the way for genomic characterisation, which will provide further insights into adaptation and evolution in human hosts by transcriptome studies and gene manipulation.

Draft genome sequence of Escherichia coli ST977: A clinical multidrug-resistant strain harbouring blaNDM-3 isolated from a bloodstream infection.

The emergence of carbapenem-resistant Escherichia coli has become a serious challenge to manage in the clinic because of multidrug resistance. Here we report the draft genome sequence of NDM-3-producing E. coli strain NT1 isolated from a bloodstream infection in China. Whole genomic DNA of E. coli strain NT1 was extracted and was sequenced using an Illumina HiSeq™ X Ten platform. The generated sequence reads were assembled using CLC Genomics Workbench. The draft genome was annotated using Rapid Annotation using Subsystem Technology (RAST). Bioinformatics analysis was further performed. The genome size was calculated at 5,353 620bp, with 5297 protein-coding sequences and the presence of genes conferring resistance to aminoglycosides, β-lactams, quinolones, macrolides, phenicols, sulphonamides, tetracycline and trimethoprim. In addition, genes encoding virulence factors were also identified. To our knowledge, this is the first report of an E. coli strain producing NDM-3 isolated from a human bloodstream infection. The genome sequence will provide valuable information to understand antibiotic resistance mechanisms and pathogenic mechanisms in this strain. Close surveillance is urgently needed to monitor the spread of NDM-3-producing isolates.

Complete Genome Sequence of Xanthomonas citri pv. anacardii Strain IBSBF2579 from Brazil.

ABSTRACTThe bacterium Xanthomonas citri pv. anacardii is the agent of angular leaf spot of the cashew tree (Anacardium occidentale L.). The complete genome sequencing of the strain IBSBF2579 was done on an Illumina HiSeq 2500 platform. The de novo assembly of the X. citri pv. anacardii strain IBSBF2579 genome yielded 133 contigs, with a size of 5,329,247 bp and a G+C content of 64.03%. The prediction was performed by GeneMarkS and the automatic annotation by Rapid Annotations using Subsystems Technology (RAST), with 4,406 identified genes.