Ceftolozane/Tazobactam Resistance and Mechanisms in Carbapenem-Nonsusceptible Pseudomonas aeruginosa

ABSTRACT This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired β-lactamases, especially metallo-β-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by β-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents. IMPORTANCE Pseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel β-lactam/β-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future β-lactams and β-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

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Different Resistance Mechanisms for Cadazolid and Linezolid in Clostridium difficile Found by Whole-Genome Sequencing Analysis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00384-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 9

Author(s):

Patrick Caspers ◽

Hans H. Locher ◽

Philippe Pfaff ◽

Sarah Diggelmann ◽

Georg Rueedi ◽

...

Keyword(s):

Clostridium Difficile ◽

Ribosomal Protein ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Cross Resistance ◽

Whole Genome ◽

Content Type

ABSTRACT Cadazolid (CDZ) is a new antibiotic currently in clinical development for the treatment of Clostridium difficile infections. CDZ interferes with the bacterial protein synthesis machinery. The aim of the present study was to identify resistance mechanisms for CDZ and compare the results to those obtained for linezolid (LZD) in C. difficile by whole-genome sequencing (WGS) of strains generated by in vitro passages and to those obtained for LZD-resistant clinical isolates. Clones of C. difficile 630 selected with CDZ during 46 passages had a maximally 4-fold increase in CDZ MIC, while the LZD MIC for clones selected with LZD increased up to 16-fold. CDZ cross-resistance with LZD was maximally 4-fold, and no cross-resistance with other antibiotics tested was observed. Our data suggest that there are different resistance mechanisms for CDZ and LZD in C. difficile. Mutations after passages with CDZ were found in rplD (ribosomal protein L4) as well as in tra and rmt, whereas similar experiments with LZD showed mutations in rplC (ribosomal protein L3), reg, and tpr, indicating different resistance mechanisms. Although high degrees of variation between the sequenced genomes of the clinical isolates were observed, the same mutation in rplC was found in two clinical isolates with high LZD MICs. No mutations were found in the 23S rRNA genes, and attempts to isolate the cfr gene from resistant clinical isolates were unsuccessful. Analysis of 50% inhibitory concentrations (IC50s) determined in in vitro transcription/translation assays performed with C. difficile cell extracts from passaged clones correlated well with the MIC values for all antibiotics tested, indicating that the ribosomal mutations are causing the resistant phenotype.

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Comparative Genome Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Sequence Type 131 Strains from Nepal and Japan

mSphere ◽

10.1128/msphere.00289-16 ◽

2016 ◽

Vol 1 (5) ◽

Cited By ~ 6

Author(s):

Tohru Miyoshi-Akiyama ◽

Jatan Bahadur Sherchan ◽

Yohei Doi ◽

Maki Nagamatsu ◽

Jeevan B. Sherchand ◽

...

Keyword(s):

Molecular Epidemiology ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sequence Type ◽

Whole Genome ◽

Asian Countries ◽

Content Type ◽

E Coli ◽

Global Spread ◽

Low Prevalence

ABSTRACT The global spread of ESBL-E. coli has been driven in large part by pandemic sequence type 131 (ST131). A recent study suggested that, within E. coli ST131, certain sublineages have disseminated worldwide with little association with their geographical origin, highlighting the complexity of the epidemiology of this pandemic clone. ST131 bacteria have also been classified into four virotypes based on the distribution of certain virulence genes. Information on virotype distribution in Asian ST131 strains is limited. We conducted whole-genome sequencing of ESBL-E. coli ST131 strains collected in Nepal and Japan, two Asian countries with a high and low prevalence of ESBL-E. coli, respectively. We systematically compared these ST131 genomes with those reported from other regions to gain insights into the molecular epidemiology of their spread and found the distinct phylogenetic characteristics of the spread of ESBL-E. coli ST131 in these two geographical areas of Asia. The global spread of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) has largely been driven by the pandemic sequence type 131 (ST131). This study aimed to determine the molecular epidemiology of their spread in two Asian countries with contrasting prevalence. We conducted whole-genome sequencing (WGS) of ESBL-E. coli ST131 strains collected prospectively from Nepal and Japan, two countries in Asia with a high and low prevalence of ESBL-E. coli, respectively. We also systematically compared these genomes with those reported from other regions using publicly available WGS data for E. coli ST131 strains. Further, we conducted phylogenetic analysis of these isolates and all genome sequence data for ST131 strains to determine sequence diversity. One hundred five unique ESBL-E. coli isolates from Nepal (February 2013 to July 2013) and 76 isolates from Japan (October 2013 to September 2014) were included. Of these isolates, 54 (51%) isolates from Nepal and 11 (14%) isolates from Japan were identified as ST131 by WGS. Phylogenetic analysis based on WGS suggested that the majority of ESBL-E. coli ST131 isolates from Nepal clustered together, whereas those from Japan were more diverse. Half of the ESBL-E. coli ST131 isolates from Japan belonged to virotype C, whereas half of the isolates from Nepal belonged to a virotype other than virotype A, B, C, D, or E (A/B/C/D/E). The dominant sublineage of E. coli ST131 was H30Rx, which was most prominent in ESBL-E. coli ST131 isolates from Nepal. Our results revealed distinct phylogenetic characteristics of ESBL-E. coli ST131 spread in the two geographical areas of Asia, indicating the involvement of multiple factors in its local spread in each region. IMPORTANCE The global spread of ESBL-E. coli has been driven in large part by pandemic sequence type 131 (ST131). A recent study suggested that, within E. coli ST131, certain sublineages have disseminated worldwide with little association with their geographical origin, highlighting the complexity of the epidemiology of this pandemic clone. ST131 bacteria have also been classified into four virotypes based on the distribution of certain virulence genes. Information on virotype distribution in Asian ST131 strains is limited. We conducted whole-genome sequencing of ESBL-E. coli ST131 strains collected in Nepal and Japan, two Asian countries with a high and low prevalence of ESBL-E. coli, respectively. We systematically compared these ST131 genomes with those reported from other regions to gain insights into the molecular epidemiology of their spread and found the distinct phylogenetic characteristics of the spread of ESBL-E. coli ST131 in these two geographical areas of Asia.

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Carriage of a Single Strain of NontoxigenicCorynebacterium diphtheriaebv. Belfanti (Corynebacterium belfantii) in Four Patients with Cystic Fibrosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00042-19 ◽

2019 ◽

Vol 57 (5) ◽

Cited By ~ 3

Author(s):

Diane Pivot ◽

Annlyse Fanton ◽

Edgar Badell-Ocando ◽

Marion Benouachkou ◽

Karine Astruc ◽

...

Keyword(s):

Cystic Fibrosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antimicrobial Agents ◽

Care Center ◽

Whole Genome ◽

Single Strain ◽

Content Type ◽

Microbiological Methods ◽

Cross Transmission

ABSTRACTCystic fibrosis (CF) patients are commonly colonized by bacterial pathogens, which can induce persistent lung inflammation and may contribute to clinical deterioration. Colonization of CF patients and cross-transmission byCorynebacterium diphtheriaehave not been reported so far. The aim of this article was to investigate the possibility of a cross-transmission ofC. diphtheriaebiovar Belfanti between four patients of a CF center.C. diphtheriaebiovar Belfanti (now formally calledC. belfantii) isolates were collected from four patients in a single CF care center over a period of 6 years and analyzed by microbiological methods and whole-genome sequencing. Epidemiological links among patients were investigated. Ten isolates were collected from 4 patients. Whole-genome sequencing of one isolate from each patient showed that a single strain was shared among them. In addition, one patient was found to have the same strain in two consecutive samplings performed 9 months apart. The strain was nontoxigenic and was susceptible to most antimicrobial agents. Ciprofloxacin resistance was observed in one patient. The idea of transmission of the strain among patients was supported by the occurrence of same-day visits to the CF center. This study demonstrated colonization of CF patients byC. diphtheriaebiovar Belfanti (C. belfantii), and the data suggest persistence and transmission of a unique strain during at least 6 years in a single CF patient care center.

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Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

Applied and Environmental Microbiology ◽

10.1128/aem.02873-15 ◽

2015 ◽

Vol 82 (2) ◽

pp. 459-466 ◽

Cited By ~ 132

Author(s):

S. Zhao ◽

G. H. Tyson ◽

Y. Chen ◽

C. Li ◽

S. Mukherjee ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Antimicrobial Agents ◽

The United States ◽

23S Rrna ◽

Whole Genome ◽

Sequencing Analysis ◽

Content Type

ABSTRACTThe objectives of this study were to identify antimicrobial resistance genotypes forCampylobacterand to evaluate the correlation between resistance phenotypes and genotypes usingin vitroantimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114Campylobacterspecies isolates (82C. coliand 32C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, includingtet(O),blaOXA-61,catA,lnu(C),aph(2″)-Ib,aph(2″)-Ic,aph(2′)-If,aph(2″)-Ig,aph(2″)-Ih,aac(6′)-Ie-aph(2″)-Ia,aac(6′)-Ie-aph(2″)-If,aac(6′)-Im,aadE,sat4,ant(6′),aad9,aph(3′)-Ic, andaph(3′)-IIIa, and mutations in two housekeeping genes (gyrAand 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs.

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Isolation, Whole-Genome Sequencing, and Annotation of Three Unclassified Antibiotic-Producing Bacteria, Enterobacter sp. Strain RIT 637, Pseudomonas sp. Strain RIT 778, and Deinococcus sp. Strain RIT 780

Microbiology Resource Announcements ◽

10.1128/mra.00863-21 ◽

2021 ◽

Vol 10 (48) ◽

Author(s):

Marissa N. Schroeter ◽

Safiya J. Gazali ◽

Anutthaman Parthasarathy ◽

Crista B. Wadsworth ◽

Renata Rezende Miranda ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Disk Diffusion ◽

Whole Genome ◽

Pseudomonas Sp ◽

Content Type

We report the isolation, whole-genome sequencing, and annotation of Enterobacter sp. strain RIT 637, Pseudomonas sp. strain RIT 778, and Deinococcus sp. strain RIT 780. Disk diffusion assays using spent medium demonstrated that all bacteria produced bactericidal compounds against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923.

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Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis andIn SilicoSerotyping of Pseudomonas aeruginosa Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.00349-16 ◽

2016 ◽

Vol 54 (7) ◽

pp. 1782-1788 ◽

Cited By ~ 17

Author(s):

Sandra Wingaard Thrane ◽

Véronique L. Taylor ◽

Ole Lund ◽

Joseph S. Lam ◽

Lars Jelsbak

Keyword(s):

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Specific Antigen ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Chronic Infections ◽

Content Type

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed thePseudomonas aeruginosaserotyper (PAst) program, which enabledin silicoserotyping ofP. aeruginosaisolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss ofin vitrotypeability often associated withP. aeruginosaisolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst,P. aeruginosaserotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.

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In vitro Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa following Treatment-emergent Resistance to Ceftolozane-tazobactam

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00084-21 ◽

2021 ◽

Author(s):

Abigail M. Rubio ◽

Ellen G. Kline ◽

Chelsea E. Jones ◽

Liang Chen ◽

Barry N. Kreiswirth ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Multidrug Resistant ◽

Cross Resistance ◽

Whole Genome ◽

In Vitro Susceptibility ◽

Before And After ◽

Increased Susceptibility

We compared the in vitro susceptibility of multidrug-resistant Pseudomonas aeruginosa isolates collected before and after treatment-emergent resistance to ceftolozane-tazobactam. Median baseline and post-exposure ceftolozane-tazobactam MICs were 2 and 64 μg/mL, respectively. Whole-genome sequencing identified treatment-emergent mutations in ampC among 79% (11/14) of paired isolates. AmpC mutations were associated with cross-resistance to ceftazidime-avibactam, but increased susceptibility to piperacillin-tazobactam and imipenem. Eighty-one percent (12/16) of ceftolozane-tazobactam resistant isolates with ampC mutations were susceptible to imipenem-relebactam.

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Chromosomally Encoded mcr-5 in Colistin-Nonsusceptible Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00679-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 24

Author(s):

Erik Snesrud ◽

Rosslyn Maybank ◽

Yoon I. Kwak ◽

Anthony R. Jones ◽

Mary K. Hinkle ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Analysis ◽

Clinical Isolates ◽

Whole Genome ◽

Broth Microdilution ◽

Colistin Resistance ◽

Content Type ◽

First Report

ABSTRACT Whole-genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was nonsusceptible to colistin by broth microdilution, and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin-nonsusceptible P. aeruginosa.

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Whole-Genome Sequencing Identifies Emergence of a Quinolone Resistance Mutation in a Case of Stenotrophomonas maltophilia Bacteremia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01723-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 7117-7120 ◽

Cited By ~ 12

Author(s):

Theodore R. Pak ◽

Deena R. Altman ◽

Oliver Attie ◽

Robert Sebra ◽

Camille L. Hamula ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Stenotrophomonas Maltophilia ◽

De Novo ◽

Resistance Mutation ◽

Resistance Mechanisms ◽

Whole Genome ◽

Multidrug Efflux ◽

Content Type ◽

Before And After

ABSTRACTWhole-genome sequences forStenotrophomonas maltophiliaserial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembledde novoand differed by one single-nucleotide variant insmeT, a repressor for multidrug efflux operonsmeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging inS. maltophiliastrains during clinical therapy.

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Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

Journal of Clinical Microbiology ◽

10.1128/jcm.01461-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 2

Author(s):

Ellen N. Kersh ◽

Cau D. Pham ◽

John R. Papp ◽

Robert Myers ◽

Richard Steece ◽

...

Keyword(s):

Public Health ◽

Antibiotic Resistance ◽

Neisseria Gonorrhoeae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Whole Genome ◽

Content Type ◽

Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

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