Dataset Information

Extensively Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem Guiana Extended Spectrum ?-Lactamase Enzymes: A Newly Emerging Threat in the United States.

ABSTRACT: Background:Treatment of serious infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 P aeruginosa resistant to all ?-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam. Methods:Isolates were sequenced using both short-read (Illumina) and long-read technology to identify resistance determinants, polymorphisms (compared with P aeruginosa PAO1), and reconstruct a phylogenetic tree. A pair of ?-lactamases, Guiana extended spectrum ?-lactamase (GES)-19 and GES-26, were cloned and expressed in a laboratory strain of Escherichia coli to examine their relative impact on resistance. Using cell lysates from E coli expressing the GES genes individually and in tandem, we determined relative rates of hydrolysis for nitrocefin and ceftazidime. Results:Two ST309 P aeruginosa clinical isolates were found to harbor the extended spectrum ?-lactamases GES-19 and GES-26 clustered in tandem on a chromosomal class 1 integron. The presence of both enzymes in E coli was associated with significantly elevated minimum inhibitory concentrations to aztreonam, cefepime, meropenem, ceftazidime/avibactam, and ceftolozane/tazobactam, compared with those expressed individually. The combination of ceftazidime/avibactam plus aztreonam was active in vitro and used to achieve cure in one patient. Phylogenetic analysis revealed ST309 P aeruginosa are closely related to MDR strains from Mexico also carrying tandem GES. Conclusions:The presence of tandem GES-19 and GES-26 is associated with resistance to all ?-lactams, including ceftolozane/tazobactam. Phylogenetic analysis suggests that ST309 P aeruginosa may be an emerging threat in the United States.

SUBMITTER: Khan A

PROVIDER: S-EPMC6602888 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Extensively Drug-Resistant <i>Pseudomonas aeruginosa</i> ST309 Harboring Tandem Guiana Extended Spectrum β-Lactamase Enzymes: A Newly Emerging Threat in the United States.

Khan Ayesha A Tran Truc T TT Rios Rafael R Hanson Blake B Shropshire William C WC Sun Zhizeng Z Diaz Lorena L Dinh An Q AQ Wanger Audrey A Ostrosky-Zeichner Luis L Palzkill Timothy T Arias Cesar A CA Miller William R WR

Open forum infectious diseases 20190606 7

<h4>Background</h4>Treatment of serious infections due to multidrug-resistant (MDR) <i>Pseudomonas aeruginosa</i> remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 <i>P aeruginosa</i> resistant to all β-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam.<h4>Methods</h4>Isolates were sequenced using both short-read (Il ...[more]

PMID: 31281867

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Project description:Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) β-lactamase-producing strains are of growing concern. Several genotypes of the GES β-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES β-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.

Project description:Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of β-lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZR) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, blaVIM, blaOXA-48, blaCMY-2, blaDHA-1, blaFOX, blaMOX, and blaACC) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6')-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZR K. pneumoniae isolates and 11 of them carried the blaCTX-M-15 gene (together with blaTEM), and the remaining isolate carried the blaSHV-106 gene. All the blaCTX-M-15 harboring isolates also contained a blaSHV gene (blaSHV-1, blaSHV-11 or blaSHV-27 variants). Both blaSHV-27 and blaSHV-106 genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (blaKPC2/3 and blaOXA-48) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.

Dataset Information

Extensively Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem Guiana Extended Spectrum ?-Lactamase Enzymes: A Newly Emerging Threat in the United States.

Publications

Extensively Drug-Resistant <i>Pseudomonas aeruginosa</i> ST309 Harboring Tandem Guiana Extended Spectrum β-Lactamase Enzymes: A Newly Emerging Threat in the United States.

Similar Datasets

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