A Rapid Antimicrobial Susceptibility Test for Klebsiella pneumoniae Using a Broth Micro-Dilution Combined with MALDI TOF MS

BACKGROUND: Chronic Suppurative Otitis Media (CSOM) causes hearing impairment and frequently occurred in low-income country where medical care and personal hygiene are poor. Staphylococcus aureus and Pseudomonas aeruginosa are the most common cause of CSOM. We investigated prevalence and antimicrobial susceptibility of S. aureus and P. aeruginosa from tubotympanic CSOM patients in tertiary hospital, Purwokerto, Indonesia in 2016-2017.METHODS: Ear swab specimens were collected from patients with tubotympanic CSOM. S. aureus and P. aeruginosa were isolated and identified by culture, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and molecular tools. Antimicrobial susceptibility testing was performed using the disk diffusion method.RESULTS: Out of ear swabs from 34 patients with tubotympanic CSOM, P. aeruginosa and S. aureus were identified in 35%patients. No Methicillin-resistant S. aureus (MRSA) strain was found from the ear swabs of the patients with tubotympanic CSOM. Bacterial identification using the MALDI-TOF MS was concordantly with culture and molecular tools. All S. aureus isolates showed full susceptibility to cefoxitin and trimethoprim-sulphamethoxazole. Resistance to tetracycline was common with only 64% of S. aureus strains being susceptible. Meanwhile, all P. aeruginosa strains were susceptible to cefepime, cetazidime, meropenem, gentamicin, and tobramycin.CONCLUSION: S. aureus and P. aeruginosa are found in patients with tubotympanic CSOM and still susceptible to different antibiotic agents. MALDI-TOF MS demonstrate rapid, accurate and robust to detect S. aureus and P. aeruginosa.KEYWORDS: Staphylococcus aureus, Pseudomonas aeruginosa, chronic tubotympanic suppurative otitis media

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Occurrence of the p019 gene in the blaKPC-harbouring plasmids: adverse clinical impact for direct tracking of KPC-producing Klebsiella pneumoniae by MALDI-TOF MS

Journal of Clinical Microbiology ◽

10.1128/jcm.00238-21 ◽

2021 ◽

Author(s):

Eva Gato ◽

Ignacio Pedro Constanso ◽

Bruno Kotska Rodiño-Janeiro ◽

Paula Guijarro-Sánchez ◽

Tyler Alioto ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Computational Analysis ◽

Direct Detection ◽

Clinical Impact ◽

Mass Peak ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Direct Tracking ◽

Geographical Locations ◽

Tof Ms

MALDI-TOF MS has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated K. pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or non carbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by WGS and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid, but it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K) and IncX3. Besides, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

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Rapid identification and antimicrobial susceptibility testing of positive blood cultures using MALDI-TOF MS and a modification of the standardised disc diffusion test: a pilot study

Journal of Clinical Pathology ◽

10.1136/jclinpath-2015-203436 ◽

2016 ◽

Vol 69 (11) ◽

pp. 1025-1032 ◽

Cited By ~ 9

Author(s):

C Fitzgerald ◽

P Stapleton ◽

E Phelan ◽

P Mulhare ◽

B Carey ◽

...

Keyword(s):

Pilot Study ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Rapid Identification ◽

Blood Cultures ◽

Disc Diffusion ◽

Diffusion Test ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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A Prospective Evaluation of Two Rapid Phenotypical Antimicrobial Susceptibility Technologies for the Diagnostic Stewardship of Sepsis

BioMed Research International ◽

10.1155/2018/6976923 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 15

Author(s):

Cesira Giordano ◽

Elena Piccoli ◽

Veronica Brucculeri ◽

Simona Barnini

Keyword(s):

Antimicrobial Susceptibility ◽

Multidrug Resistant ◽

Rapid Identification ◽

Blood Cultures ◽

Gram Positive ◽

Gram Negative ◽

Maldi Tof Ms ◽

Gram Positive Bacteria ◽

Maldi Tof ◽

Tof Ms

Rapid identification of bloodstream pathogens by MALDI-TOF MS and the recently introduced rapid antimicrobial susceptibility testing (rAST) directly from positive blood cultures allow clinicians to promptly achieve a targeted therapy, especially for multidrug resistant microorganisms. In the present study, we propose a comparison between phenotypical rASTs performed in light-scattering technology (Alfred 60AST, Alifax®) and fluorescencein situhybridization (Pheno™, Accelerate) directly from positive blood cultures, providing results in 4–7 hours. Blood samples from 67 patients admitted to the Azienda Ospedaliero-Universitaria Pisana were analyzed. After the direct MALDI-TOF MS identification, the rAST was performed at the same time both on Alfred 60AST and Pheno. Alfred 60AST provided qualitative results, interpreted in terms of clinical categories (SIR). Pheno provided identification and MIC values for each antibiotic tested. Results were compared to the broth microdilution assay (SensiTitre™, Thermo Fisher Scientific), according to EUCAST rules. Using Alfred 60AST, an agreement was reached, 91.1% for Gram-negative and 95.7% for Gram-positive bacteria, while using Pheno, the agreement was 90.6% for Gram-negative and 100% for Gram-positive bacteria. Both methods provided reliable results; Alfred 60AST combined with MALDI-TOF MS proved itself faster and cheaper. Pheno provided identification and MIC determination in a single test and, although more expensive, may be useful whenever MIC value is necessary and where MALDI-TOF MS is not present.

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Direct antimicrobial susceptibility tests of bacteria and yeasts from positive blood cultures by using serum separator gel tubes and MALDI–TOF MS

Journal of Microbiological Methods ◽

10.1016/j.mimet.2018.12.011 ◽

2019 ◽

Vol 157 ◽

pp. 16-20 ◽

Cited By ~ 6

Author(s):

Shenghai Wu ◽

Jie Xu ◽

Chunning Qiu ◽

Lihui Xu ◽

Qiong Chen ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Blood Cultures ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Antimicrobial Susceptibility Tests ◽

Susceptibility Tests ◽

Tof Ms

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MALDI-TOF MS identification and antimicrobial susceptibility testing directly from positive enrichment broth

Journal of Microbiological Methods ◽

10.1016/j.mimet.2017.07.012 ◽

2017 ◽

Vol 141 ◽

pp. 32-34 ◽

Cited By ~ 3

Author(s):

Marie Tré-Hardy ◽

Barbara Lambert ◽

Noémie Despas ◽

Florian Bressant ◽

Clémentine Laurenzano ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Antimicrobial Susceptibility Testing ◽

Enrichment Broth ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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MALDI-TOF Mass Spectrometry-Based Optochin Susceptibility Testing for Differentiation of Streptococcus pneumoniae from other Streptococcus mitis Group Streptococci

Microorganisms ◽

10.3390/microorganisms9102010 ◽

2021 ◽

Vol 9 (10) ◽

pp. 2010

Author(s):

Ilka D. Nix ◽

Evgeny A. Idelevich ◽

Andreas Schlattmann ◽

Katrin Sparbier ◽

Markus Kostrzewa ◽

...

Keyword(s):

Mass Spectrometry ◽

Streptococcus Pneumoniae ◽

Incubation Time ◽

Susceptibility Test ◽

Test Accuracy ◽

Streptococcus Mitis ◽

Pathogenic Potential ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL; correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study; however, confirmation and further improvements are warranted.

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