Validation of MALDI-TOF for the early detection of the ST175 high-risk clone of Pseudomonas aeruginosa in clinical isolates belonging to a Spanish nationwide multicenter study

Mulet, Xavier; Fernández-Esgueva, Marta; Norte, Cristina; Zamorano, Laura; Barrio-Tofiño, Ester del; Oliver, Antonio

doi:10.1016/j.eimc.2020.05.022

Cited by 6 publications

(4 citation statements)

References 13 publications

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“…Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) states an advanced technology and owns a very good application prospects in identifying P. aeruginosa ( Wilhelm et al, 2021 ). MALDI-TOF-MS was used to accurately and rapidly identify the five high-risk clones of P. aeruginosa sequence ST111, ST175, ST235, ST253, and ST395, also was applied in P. aeruginosa drug resistance analysis such as carbapenemase ( Mulet et al, 2021 ). MALDI-TOF-MS exhibits limited resolving power, and therefore does not supply sequence-based ID necessarily; microbial ID using MALDI-TOF-MS is based on spectral fingerprint patterns rather than the identity of each spectral peak ( Ayhan et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis

Wang

Ai-ming

et al. 2022

Front. Microbiol.

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Mining novel specific molecular targets and establishing efficient identification methods are significant for detecting Pseudomonas aeruginosa, which can enable P. aeruginosa tracing in food and water. Pangenome analysis was used to analyze the whole genomic sequences of 2017 strains (including 1,000 P. aeruginosa strains and 1,017 other common foodborne pathogen strains) downloaded from gene databases to obtain novel species-specific genes, yielding a total of 11 such genes. Four novel target genes, UCBPP-PA14_00095, UCBPP-PA14_03237, UCBPP-PA14_04976, and UCBPP-PA14_03627, were selected for use, which had 100% coverage in the target strain and were not present in nontarget bacteria. PCR primers (PA1, PA2, PA3, and PA4) and qPCR primers (PA12, PA13, PA14, and PA15) were designed based on these target genes to establish detection methods. For the PCR primer set, the minimum detection limit for DNA was 65.4 fg/μl, which was observed for primer set PA2 of the UCBPP-PA14_03237 gene. The detection limit in pure culture without pre-enrichment was 105 colony-forming units (CFU)/ml for primer set PA1, 103 CFU/ml for primer set PA2, and 104 CFU/ml for primer set PA3 and primer set PA4. Then, qPCR standard curves were established based on the novel species-specific targets. The standard curves showed perfect linear correlations, with R2 values of 0.9901 for primer set PA12, 0.9915 for primer set PA13, 0.9924 for primer set PA14, and 0.9935 for primer set PA15. The minimum detection limit of the real-time PCR (qPCR) assay was 102 CFU/ml for pure cultures of P. aeruginosa. Compared with the endpoint PCR and traditional culture methods, the qPCR assay was more sensitive by one or two orders of magnitude. The feasibility of these methods was satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 29 ready-to-eat vegetable samples and was almost consistent with that of the national standard detection method. The developed assays can be applied for rapid screening and detection of pathogenic P. aeruginosa, providing accurate results to inform effective monitoring measures in order to improve microbiological safety.

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Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis

Wang

Ai-ming

et al. 2022

Front. Microbiol.

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“…Moreover, the 3R rules [ 36 ] (Hubrecht and Carter, 2019) prevent us from increasing the numbers of animals to test the same hypothesis in male mice. As strengths of the study, the chosen isolates from the GEMARA/REIPI collection [ 23 ], both for the in vitro and in vivo studies, are representative of high-risk clones and widespread acquired carbapenemases [ 15 , 37 , 38 , 39 ].…”

Section: Discussionmentioning

confidence: 99%

Carbapenem Combinations for Infections Caused by Carbapenemase-Producing Pseudomonas aeruginosa: Experimental In Vitro and In Vivo Analysis

et al. 2022

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In the context of difficult-to-treat carbapenem-resistant Pseudomonas aeruginosa infections, we evaluated imipenem, meropenem, and doripenem combinations against eleven carbapenemase-producing P. aeruginosa isolates. According to the widespread global distribution of high-risk clones and carbapenemases, four representative isolates were selected: ST175 (OXA-2/VIM-20), ST175 (VIM-2), ST235 (GES-5), and ST111 (IMP-33), for efficacy studies using a sepsis murine model. Minimum inhibitory concentration (mg/L) ranges were 64–256 for imipenem and 16–128 for meropenem and doripenem. In vitro, imipenem plus meropenem was synergistic against 72% of isolates and doripenem plus meropenem or imipenem against 55% and 45%, respectively. All combinations were synergistic against the ST175, ST235, and ST155 clones. In vivo, meropenem diminished the spleen and blood bacterial concentrations of four and three isolates, respectively, with better efficacy than imipenem or doripenem. The combinations did not show efficacy compared with the more active monotherapies, except for imipenem plus meropenem, which reduced the ST235 bacterial spleen concentration. Mortality decreased with imipenem plus meropenem or doripenem for the ST175 isolate. Results suggest that carbapenem combinations are not an alternative for severe infections by carbapenemase-producing P. aeruginosa. Meropenem monotherapy showed in vivo efficacy despite its high MIC, probably because its dosage allowed a sufficient antimicrobial exposure at the infection sites.

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“…MALDI‐TOF MS has been used to identify high‐risk clones of P. aeruginosa with a sensitivity and specificity of 97.1% and 99.4%, respectively (Mulet et al, 2021). Culture using Pseudomonas ‐selective media has shown a high sensitivity (98–100%) but low specificity ranging between 40% and 72% (Weiser et al, 2014).…”

Section: Assessmentmentioning

confidence: 99%

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): antimicrobial‐resistant Pseudomonas aeruginosa in dogs and cats

Nielsen¹,

Bicout²,

Calistri³

et al. 2022

EFS2

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Pseudomonas aeruginosa (P. aeruginosa) was identified among the most relevant antimicrobial‐resistant (AMR) bacteria in the EU for dogs and cats in a previous scientific opinion. Thus, it has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9, and Article 8 for listing animal species related to the bacterium. The assessment has been performed following a methodology previously published. The outcome is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with uncertain outcome. According to the assessment here performed, it is uncertain whether AMR P. aeruginosa can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (33–90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that the bacterium does not meet the criteria in Sections 1, 2, 3 and 4 (Categories A, B, C and D; 0–5%, 1–5%, 5–33% and 5–33% probability of meeting the criteria, respectively) and the AHAW Panel was uncertain whether it meets the criteria in Section 5 (Category E, 33–90% probability of meeting the criteria). The animal species to be listed for AMR P. aeruginosa according to Article 8 criteria are mainly dogs and cats.

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Validation of MALDI-TOF for the early detection of the ST175 high-risk clone of Pseudomonas aeruginosa in clinical isolates belonging to a Spanish nationwide multicenter study

Cited by 6 publications

References 13 publications

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis

Carbapenem Combinations for Infections Caused by Carbapenemase-Producing Pseudomonas aeruginosa: Experimental In Vitro and In Vivo Analysis

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): antimicrobial‐resistant Pseudomonas aeruginosa in dogs and cats

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