scholarly journals Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Mahjabeen Khan ◽  
Stephen Summers ◽  
Scott A Rice ◽  
Fiona Stapleton ◽  
Mark D P Willcox ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Genes ◽  
Empirical Therapy ◽  
Illumina Miseq ◽  
Whole Genome Sequence ◽  
Fluoroquinolone Resistance ◽  
Beta Lactam ◽  
Long Reads ◽  
Long Read ◽  
Class I Integrons

AbstractFluroquinolones are widely used as an empirical therapy for pseudomonal ocular infections. Based on increasing reports on acquired fluroquinolone resistance genes in clinical isolates of Pseudomonas aeruginosa, we investigated 33 strains of P. aeruginosa isolated from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018 to understand the prevalence of acquired fluroquinolone resistance genes in ocular isolates and to assess whether the possession of those genes was associated with fluoroquinolone susceptibility. We obtained the whole genome sequence of 33 isolates using Illumina MiSeq platform and investigated the prevalence of two fluoroquinolone resistance genes crpP and qnrVC1. To examine the associated mobile genetic elements of qnrVC1 positive strains, we obtained long read sequences using Oxford Nanopore MinION and performed hybrid assembly to combine long reads with Illumina short sequence reads. We further assessed mutations in QRDRs and antibiotic susceptibilities to ciprofloxacin, levofloxacin and moxifloxacin to examine the association between resistance genes and phenotype. Twenty strains possessed crpP in genetic islands characterised by possession of integrative conjugative elements. The qnrVC1 gene was carried by four isolates on class I integrons and Tn3 transposons along with aminoglycoside and beta-lactam resistance genes. We did not observe any evidence of plasmids carrying fluroquinolone resistance genes. Resistance to fluroquinolones was observed in those strains which possessed crpP, qnrVC1 and that had QRDRs mutations. The presence of crpP was not a sole cause of fluroquinolone resistance.

scholarly journals 606. Identification and Characterization of HMB-2, a Novel Metallo-β-Lactamase in a Pseudomonas aeruginosa Isolate

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S283-S284
Author(s):  
Wenming Zhu ◽  
Gillian A McAllister ◽  
Maria Jose Machado ◽  
Davina Campbell ◽  
Maria Karlsson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Illumina Miseq ◽  
Multidrug Resistant ◽  
Urine Specimen ◽  
Emerging Infections ◽  
E Coli ◽  
Long Read ◽  
Protein Sequence Identity ◽  
Topo Ii ◽  
Lactamase Gene

Abstract Background Carbapenemases, a global health threat, are a diverse group of β-lactamases active against cephalosporins and carbapenems, which are often last resort treatments for multidrug-resistant gram-negative infections. The most common carbapenemases reported among Pseudomonas aeruginosa are metallo-β-lactamase (MBLs). We describe a novel MBL (designated HMB-2) identified in a P. aeruginosa isolate from a urine specimen collected in 2015 as part of CDC’s Emerging Infections Program. Methods We performed antimicrobial susceptibility testing (AST) by broth microdilution, real-time PCR to screen for common carbapenemases (IMP, KPC, NDM, VIM, and OXA-48), and modified carbapenem inactivation method (mCIM) to test for carbapenemase production. The isolate underwent whole-genome sequencing (WGS) using Illumina MiSeq and PacBio RS II (Pacific Biosciences) platforms. Long read sequences were polished using Quiver and corrected by Pilon utilizing Illumina reads. We further characterized a putative novel MBL identified in WGS data by amplifying and cloning the gene into the pCR2.1-TOPO II vector (Invitrogen), which was then sub-cloned into a pET21 expression vector (Sigma–Aldrich). The resulting hmb2+ pET21 plasmid was transformed into a susceptible Escherichia coli for AST, including the imipenem-EDTA method to confirm MBL activity. Results The isolate displayed resistance to carbapenems and demonstrated phenotypic carbapenemase activity (mCIM positive), but was negative for carbapenemase genes by PCR. WGS analyses identified a putative MBL gene located on the chromosome. The gene shared 98% DNA and protein sequence identity with an MBL reported in 2016 in a P. aeruginosa isolate from Germany (HMB-1) and thus was named hmb-2. The cloned hmb-2 gene conferred resistance to carbapenems (meropenem and ertapenem) and third-generation cephalosporins (cefotaxime and ceftazidime) in transformed E. coli. The Minimum Inhibitory Concentrationratio for the imipenem-EDTA method was ≥4. Conclusion A putative, novel β-lactamase gene, blaHMB-2, was identified and cloned. The imipenem-EDTA results indicated that HMB-2 is an MBL. This discovery underscores the important role WGS plays in identifying new mechanisms of antimicrobial resistance. Disclosures All authors: No reported disclosures.

scholarly journals Antibiogram of the Common Uropathogenic Bacteria among Yemeni Patients in Sana’a City: A Recent Report

2018 ◽  
pp. 1-10 ◽  
Author(s):  
Hafez Alsumairy ◽  
Tawfique K. AlZubiery ◽  
Talal Alharazi ◽  
Mufeed Baddah ◽  
Adel Al-Zubeiry
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Empirical Therapy ◽  
High Sensitivity ◽  
Resistance Rate ◽  
Beta Lactam ◽  
Cross Sectional ◽  
Sensitivity Pattern ◽  
Uropathogenic Bacteria

Aims: This study aimed to identify the prevalence and antimicrobial susceptibility of the commonly isolated uropathogens in Sana’a city, Yemen. Study Design:  A cross-sectional and descriptive study. Place and Duration of Study: The study was carried out at the hospitals and clinics of Sana'a city, Yemen between October 2016 and March 2017. Methodology: Clean-catch mid-stream urine samples were collected to detect the most common uropathogenic bacteria and their antibiotic susceptibility using Kirby Bauer standardized method. Results: Urine cultures yielded 170 significant bacterial growths of uropathogens. Escherichia coli was the most often isolated pathogen (43.5%), followed by Klebsiella pneumoniae (24.7%), Pseudomonas aeruginosa (20.0%) and Staphylococcus aureus (11.8%). The overall sensitivity was high to an excellent pattern for Carbapenems, Nitrofurantoin, Amikacin, and Piperacillin-Tazobactam. Escherichia coli shows an excellent sensitivity (88%) for Nitrofurantoin and Imipenem, followed by (85%) Ertapenem. Pseudomonas aeruginosa exhibited moderate resistance to Carbapenems, Moxifloxacin, and Piperacillin-Tazobactam in this study. Staphylococcus aureus was more vulnerable to all Quinolones except Nalidixic acid and it displays a high sensitivity pattern, 90% for both Nitrofurantoin and Gentamicin, 83% for Penicillin, 80% for both Minocycline. Antibiogram of isolated organisms revealed that there was resistance to two and more antimicrobials. Conclusion: In this study, we observe a high resistance rates to Beta-lactam, Quinolones, and Macrolides antibiotics. Nevertheless, most uropathogenic isolates were still sensitive to Nitrofurantoin, Imipenem, Ertapenem, and Amikacin, they considered as a proper antibiotics for empirical therapy of UTIs. Establishment of antibiogram of locally isolated organisms is necessary to avoid indiscriminating use of antibiotic and to decrease the resistance rate in our community.

scholarly journals De-novo Assembly of Limnospira fusiformis Using Ultra-Long Reads

2021 ◽  
Vol 12 ◽  
Author(s):  
McKenna Hicks ◽  
Thuy-Khanh Tran-Dao ◽  
Logan Mulroney ◽  
David L. Bernick
Keyword(s):  
Phylogenetic Analysis ◽  
Type Strain ◽  
Reference Genome ◽  
De Novo ◽  
Illumina Miseq ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Rdna Analysis

The Limnospira genus is a recently established clade that is economically important due to its worldwide use in biotechnology and agriculture. This genus includes organisms that were reclassified from Arthrospira, which are commercially marketed as “Spirulina.” Limnospira are photoautotrophic organisms that are widely used for research in nutrition, medicine, bioremediation, and biomanufacturing. Despite its widespread use, there is no closed genome for the Limnospira genus, and no reference genome for the type strain, Limnospira fusiformis. In this work, the L. fusiformis genome was sequenced using Oxford Nanopore Technologies MinION and assembled using only ultra-long reads (>35 kb). This assembly was polished with Illumina MiSeq reads sourced from an axenic L. fusiformis culture; axenicity was verified via microscopy and rDNA analysis. Ultra-long read sequencing resulted in a 6.42 Mb closed genome assembled as a single contig with no plasmid. Phylogenetic analysis placed L. fusiformis in the Limnospira clade; some Arthrospira were also placed in this clade, suggesting a misclassification of these strains. This work provides a fully closed and accurate reference genome for the economically important type strain, L. fusiformis. We also present a rapid axenicity method to isolate L. fusiformis. These contributions enable future biotechnological development of L. fusiformis by way of genetic engineering.

scholarly journals Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa

2020 ◽  
Vol 85 ◽  
pp. 104574
Author(s):  
Mahjabeen Khan ◽  
Stephen Summers ◽  
Scott A. Rice ◽  
Fiona Stapleton ◽  
Mark D.P. Willcox ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Genes ◽  
Fluoroquinolone Resistance

scholarly journals Surveillance of potential pathogens and antibiotic resistance in wastewater and surface water from Boston, USA and Vellore, India using long-read metagenomic sequencing

2021 ◽  
Author(s):  
Erica R Fuhrmeister ◽  
Lee E Voth-Gaeddert ◽  
Angeline Metilda ◽  
Albert Tai ◽  
Rebecca E Batorsky ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Surface Water ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Metagenomic Sequencing ◽  
Antibiotic Resistant ◽  
Short Read ◽  
Long Reads ◽  
Long Read

Environmental sampling (wastewater) could be an efficient surveillance strategy to capture global emerging trends in the spread of antibiotic resistance. Long-read DNA sequencing can resolve the genetic context of antibiotic resistance genes (ARGs) and is a promising tool for non- culture-based monitoring of antibiotic-resistant pathogens and ARGs in environmental samples, but has not been rigorously validated against conventional methods. We tested long-read sequencing using the portable Nanopore MinION for surveying pathogens, ARGs, and antibiotic- resistant pathogens in municipal wastewater, hospital wastewater, and surface water collected from Boston, USA and Vellore, India. We compared detection of enteric pathogens by assembly of long reads, with and without short-read polishing, and unassembled raw long reads for ARGs to multiplex real-time PCR. Using real-time PCR as a benchmark, long-read metagenomics was 49% sensitive and 75% specific at pathogen detection in assembled contigs, and 16% sensitive and 100% specific at detecting 28 clinically relevant resistance genes in raw long reads. Short- read polishing did not substantially improve pathogen identification or impact ARG identification in the assembled contigs, demonstrating that short-read polishing is not required, which greatly reduces costs. The high specificity of ARG detection supports portable long-read sequencing as a valuable tool to profile ARGs and antibiotic-resistant pathogens for environmental surveillance programs.

scholarly journals Changes to the resistome of Pseudomonas aeruginosa clone ST308 associated with corneal infection over time

2020 ◽  
Author(s):  
Mahjabeen Khan ◽  
Mark D P Willcox ◽  
Scott A Rice ◽  
Savitri Sharma ◽  
Fiona Stapleton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Genes ◽  
Acquired Resistance ◽  
List Type ◽  
Beta Lactam ◽  
Class 1 Integron ◽  
Insertions And Deletions ◽  
Resistance To Antibiotics ◽  
Class 1 ◽  
Over Time

AbstractObjectivesThis study compared the resistomes of isolates of Pseudomonas aeruginosa clone ST308 from 2018 and 1997 from India.MethodsTwo ocular clonal type ST308 isolates of Pseudomonas aeruginosa (198 and 219) isolated in 2018 and five historical isolates (31, 32, 33, 35 and 37) isolated in 1997 at the LV Prasad Eye Institute in India were analysed for their susceptibilities to ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. DNA was extracted using the DNeasy® Blood and Tissue. Paired-end library was prepared using Nextera XT DNA library preparation kit. Libraries were sequenced on Illumina® MiSeq bench top sequencer generating 300 bp paired-end reads. Spades v3.12.0 was used for assembly, Resfinder v3.1. for acquired resistance genes and Snippy V2 for variants calling. Integron finder v1.5.1 was used to identify the integrons present in the genomes.ResultsThe recent isolate 219 was resistant to all tested antibiotics except polymyxin while isolate 198 was resistant to ciprofloxacin, levofloxacin, gentamicin and tobramycin. Among historical isolates five were resistant to gentamicin, tobramycin and ciprofloxacin, four were resistant to levofloxacin while two were resistant to polymyxin. Twenty-four acquired resistance genes were present in the 2018 isolates compared to 11 in the historical isolates. All isolates contained the following genes encoding for aminoglycoside aph(6)-Id, aph(3′)-lIb, aph(3″)-Ib), beta-lactam (blaPAO), tetracycline (tet(G)), fosfomycin (fosA), chloramphenicol (catB7), sulphonamide (sul1), quaternary ammonium (qacEdelta1) and fluoroquinolone (crpP) resistance. Isolate 198 possessed aph(3′)-VI, rmtD2, qnrVC1, blaOXA-488, blaPME-1, while 219 possessed aadA1, rmtB, aac(6′)-Ib-cr, blaTEM-1B, blaVIM-2, mph(E), mph(A), msr(E). In the isolate 219 genes blaTEM-1b, blaVIM-2, sul1, qnrvc1, rmtB and aadA1 were carried on class 1 integron. While an incomplete class 1 integron was also found in isolate 198 which was located on the genome where gene rmtB, blaPME-1, qnrVC1 and sul1 genes were positioned. There were no notable differences in the number of single nucleotide polymorphisms, but recent isolates carried more insertions and deletions in their genes.ConclusionP. aeruginosa ocular clonal isolates have changed over time, with strains acquiring genes and having more insertions and deletions in their chromosomal genes that confirm resistance to antibiotics.HighlightsRecent clonal ocular isolates of Pseudomonas aeruginosa from India have acquired a number of resistance genes compared to historical clonesConsequently, resistance to antibiotics particularly fluoroquinolones in recent clones of P. aeruginosa appears to have increased.The acquired resistance genes found in the recent P. aeruginosa isolates were related to mobile genetic elements.

scholarly journals The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

2021 ◽  
Vol 66 (2) ◽  
Author(s):  
Anton Shikov ◽  
Viktoriya Tsay ◽  
Mikhail Fedyakov ◽  
Yuri Eismont ◽  
Alena Rudnik ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
False Negative ◽  
Variant Calling ◽  
Illumina Miseq ◽  
Routine Practice ◽  
Jaccard Coefficient ◽  
High Coverage ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

The emergence of long-read sequencing technologies has made a revolutionary step in genome biology and medicine. However, long reads are characterized by a relatively high error rate, impairing their usage for variant calling as a part of routine practice. Thus, we here examine different popular variant callers on long-read sequences of the human mitochondrial genome, convenient in terms of small size and easily obtained high coverage. The sequencing of mitochondrial DNA from 8 patients was conducted via Illumina (MiSeq) and the Oxford Nanopore platform (MinION), with the former utilized as a gold standard when evaluating variant calling’s accuracy. We used a conventional GATK3-BWA-based pipeline for paired-end reads and Guppy basecaller coupled with minimap2 for MinION data, respectively. We then compared the outputs of Clairvoyante, Nanopolish, GATK3, Longshot, DeepVariant, and Varscan tools applied on long-read alignments by analyzing false-positive and false-negative rates. While for most callers, raw signals represented false positives due to homopolymeric errors, Nanopolish demonstrated both high similarity (Jaccard coefficient of 0.82) and a comparable number of calls with the Illumina data (140 vs. 154) with the best performance according to AUC (area under ROC curve, 0.953) as well. In sum, our results, despite being obtained from a small dataset, provide evidence that sufficient coverage coupled with an optimal pipeline could make long reads of mitochondrial DNA applicable for variant calling.

scholarly journals Long-read sequencing revealed cooccurrence, host range, and potential mobility of antibiotic resistome in cow feces

2021 ◽  
Vol 118 (25) ◽  
pp. e2024464118
Author(s):  
Xun Qian ◽  
Santosh Gunturu ◽  
Wei Sun ◽  
James R. Cole ◽  
Bo Norby ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Mercury Resistance ◽  
Circular Chromosome ◽  
Antibiotic Resistant ◽  
Integrative Conjugative Element ◽  
Long Reads ◽  
Physical Linkage ◽  
Long Read ◽  
Current Sequence

While it is well recognized that the environmental resistome is global, diverse, and augmented by human activities, it has been difficult to assess risk because of the inability to culture many environmental organisms, and it is difficult to evaluate risk from current sequence-based environmental methods. The four most important criteria to determine risk are whether the antibiotic-resistance genes (ARGs) are a complete, potentially functional complement; if they are linked with other resistances; whether they are mobile; and the identity of their host. Long-read sequencing fills this important gap between culture and short sequence-based methods. To address these criteria, we collected feces from a ceftiofur-treated cow, enriched the samples in the presence of antibiotics to favor ARG functionality, and sequenced long reads using Nanopore and PacBio technologies. Multidrug-resistance genes comprised 58% of resistome abundance, but only 0.8% of them were plasmid associated; fluroquinolone-, aminoglycoside-, macrolide-lincosamide-streptogramin (MLS)-, and β-lactam–resistance genes accounted for 2.7 to 12.3% of resistome abundance but with 19 to 78% located on plasmids. A variety of plasmid types were assembled, some of which share low similarity to plasmids in current databases. Enterobacteriaceae were dominant hosts of antibiotic-resistant plasmids; physical linkage of extended-spectrum β-lactamase genes (CTX-M, TEM, CMY, and CARB) was largely found with aminoglycoside-, MLS-, tetracycline-, trimethoprim-, phenicol-, sulfonamide-, and mercury-resistance genes. A draft circular chromosome of Vagococcus lutrae was assembled; it carries MLS-, tetracycline- (including tetM and tetL on an integrative conjugative element), and trimethoprim-resistance genes flanked by many transposase genes and insertion sequences, implying that they remain transferrable.

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