The E. coli MHT18 and MHX43 isolates and P. mirabilis MH438 and MH42F isolates were isolated by the Clinical Microbiology Service at Memorial Hospital, Memorial Sloan Kettering Cancer Center. Antimicrobial susceptibility testing of these isolates was performed using a Neg MIC 43 panel on a Microscan System (Beckman Coulter) by the Clinical Microbiology Service at Memorial Hospital. The K. pneumoniae MH258 and MH189 isolates were previously described (Xiong et al., 2015 (link)). These isolates of K. pneumoniae, E. coli, and P. mirabilis were routinely grown overnight in LB broth (BD) with 100 µg/ml ampicillin (Fisher Scientific) at 37°C under aerobic conditions. E. coli Nissle 1917 and the isogenic E. coli Nissle 1917 cydAB/napA/narG/narZ strain were routinely grown in LB broth as previously described (Byndloss et al., 2017 (link)).
Microscan system
Manufactured by Beckman Coulter
Sourced in United States, France
The MicroScan system is a fully automated microbiology platform designed for the identification and antimicrobial susceptibility testing of a wide range of bacteria and yeast isolates. The system utilizes a combination of biochemical and turbidimetric methods to provide rapid and accurate results.
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Lab products found in correlation
Vitek 2 automated system, by bioMérieux (2 mentions)
Xpert vana vanb cartridges, by Cepheid (2 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Lb broth, by BD (1 mentions)
Platelia aspergillus eia, by Bio-Rad (1 mentions)
Maldi tof ms system, by Bruker (1 mentions)
Escherichia coli dh5α cells, by Thermo Fisher Scientific (1 mentions)
E test strip, by Liofilchem (1 mentions)
Pureyield plasmid midiprep system, by Promega (1 mentions)
Etest, by bioMérieux (1 mentions)
Phoenix nmic id 94, by BD (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
Bact alert 3d, by bioMérieux (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
10 protocols using microscan system
1
Antimicrobial Susceptibility of Clinical Isolates
2
MALDI-TOF MS for Microbial Identification
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Isolated colonies from biological samples were identified by the Matrix-Assisted Laser Desorption Ionization–Time Of Flight Mass Spectrometry (MALDI-TOF MS) system (Bruker Daltonik GmbH, Bremen, Germany). Antimicrobial susceptibility tests were performed with Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France) or with the MicroScan system (Beckman Coulter, Brea, CA, USA), according to the manufacturer’s instructions.
Fungal cultures were incubated for 7 days at 30 °C on Sabouraud selective media, whereas GM test in serum and TBA was performed according to manufacturer’s instructions (Platelia Aspergillus EIA, Bio-Rad).
Fungal cultures were incubated for 7 days at 30 °C on Sabouraud selective media, whereas GM test in serum and TBA was performed according to manufacturer’s instructions (Platelia Aspergillus EIA, Bio-Rad).
3
Biofilm-producing Bloodstream Pathogens
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We retrospectively selected a total of 248 strains, 157 of which were Staphylococcus aureus (Supplementary Table S1 ) and 91 Candida albicans (Supplementary Table S2 ). Strains were isolated from blood cultures of patients with bloodstream infections between 2012 and 2015. All isolates were previously identified by MALDI-TOF MS and their antibiotic susceptibility profile was determined using the broth microdilution method Microscan® System (Beckman-Coulter, CA, United States) applying EUCAST (2021) criteria. Moreover, the isolates were also tested for biofilm production. Then, they were kept at −80°C in skimmed milk for further analysis.
We selected S. aureus and C. albicans strains isolated from patients with bacteremia, as they are the most virulent and difficult-to-treat microorganisms in which biofilm production in catheter surface is almost the cause of the bacteremia.
We selected S. aureus and C. albicans strains isolated from patients with bacteremia, as they are the most virulent and difficult-to-treat microorganisms in which biofilm production in catheter surface is almost the cause of the bacteremia.
4
Plasmid Profiling of KPC-producing Klebsiella
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Purified plasmid DNA was obtained from overnight 50-mL LB liquid broth cultures of K. pneumoniae isolates. Plasmid extraction was performed using the PureYield plasmid midiprep system (Promega Italia Srl). Plasmid DNA was transformed into chemically competent Escherichia coli DH5-α cells (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), selecting transformants on LB agar plates containing ceftazidime (CAZ, 6 mg/L). After 24 h, colonies were screened for blaKPC using the KPCFW and KPCRV primer pair, as previously described (13 (link)).
The CAZ and MEM MICs of the KPC-positiveE. coli DH5α transformants were tested using microdilution (MicroScan system; Beckman Coulter Inc.). The CZA MICs were determined using Etest strips (Liofilchem).
The plasmid content of transformants showing different CZA and MEM resistance was sequenced using ONT. To confirm the position of blaKPC-31 and blaKPC-3 at the Tn4401 copies flanking the traS and traN genes, respectively, PCR was performed with the KPCINT/TraNRV and KPCINT/TraSFW primer pairs, followed by nested PCR performed with the KPCINT and KPCRV primer pair. The blaKPC amplicons were sequenced using the Sanger method with both the KPCINT and KPCRV primers (see Table S1 in the supplemental material).
The CAZ and MEM MICs of the KPC-positive
The plasmid content of transformants showing different CZA and MEM resistance was sequenced using ONT. To confirm the position of blaKPC-31 and blaKPC-3 at the Tn4401 copies flanking the traS and traN genes, respectively, PCR was performed with the KPCINT/TraNRV and KPCINT/TraSFW primer pairs, followed by nested PCR performed with the KPCINT and KPCRV primer pair. The blaKPC amplicons were sequenced using the Sanger method with both the KPCINT and KPCRV primers (see Table S1 in the supplemental material).
5
Antimicrobial Susceptibility Profiling
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The susceptibility to penicillin, ampicillin, erythromycin, clindamycin, gentamicin, streptomycin, tetracycline, ciprofloxacin, levofloxacin, linezolid, vancomycin, teicoplanin, daptomycin, and sulfamethoxazole-trimethoprim was studied using the MicroScan® system (Beckmann Coulter, Nyon, Switzerland). The MICs to chloramphenicol, florfenicol, kanamycin, and rifampicin were determined by broth macrodilution using Staphylococcus aureus ATCC® 29213 and E. faecalis ATCC® 29212 (Manassas, VA, USA) as quality controls, and to linezolid and tedizolid by E-test® (bioMérieux, Durham, NC, USA). The antimicrobial susceptibility testing was performed, depending on the hospital, according to CLSI [21 ] or EUCAST [22 ] standards. The CLSI criteria [21 ] were used in the case of linezolid for all enterococcal isolates.
6
National Antimicrobial Resistance Trends in Korea
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Antimicrobial resistance data was collected from 31 secondary or tertiary hospitals; 16 of these hospitals reported annually from 2013 to 2015. These 16 hospitals (620–1,356 beds) covered eight major cities and provinces in Korea, representing national antimicrobial resistance trends. The hospitals used their own hospital system or WHONET software [6 (link)] and duplicate isolates were excluded from the analysis. Antimicrobial susceptibility tests were performed by using disk diffusion or modified broth microdilution tests with the VITEK 2 automated system (bioMérieux, Marcy l'Etoile, France) or the MicroScan system (Beckman Coulter, Brea, CA, USA) according to the CLSI guidelines [7 ]. If the CLSI guideline threshold was not available, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) threshold was applied for the determination of susceptibility. To minimize size difference bias between the hospitals, data consisting of less than 20 isolates of an organism were excluded; resistance rates were calculated as the median and range because of the limited number of hospitals. The antimicrobial resistance of gram-negative bacilli was compared with previous results from 2004 to determine long-term trend changes [8 9 10 ].
7
Fluoroquinolone Resistance Determination
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The phenotypic resistance to ciprofloxacin and levofloxacin was determined using an automated MicroScan system (Beckman Coulter, Porterville, CA, USA) and BD Phoenix NMIC/ID-94 (Becton Dickinson, Franklin Lakes, NJ, USA) through the minimum inhibitory concentration (MIC) method following the recommendations of the CLSI [17 ]. The resistance to enrofloxacin (5 mg) was determined by the Kirby–Bauer disk diffusion susceptibility test. A bacterial suspension was spread in Mueller-Hinton agar (Oxoid, Wesel, Germany) according to the McFarland turbidity scale of 0.5; then, bacterial growth inhibition was evaluated at 37°C for 18 h according to the CLSI guidelines [17 ].
8
Blood Culture Diagnostic Protocol
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Two or three pairs of BC bottles, for aerobes or anaerobes, were incubated in a BacT/Alert 3D (bioMérieux, Durham, NC, USA) or BACTEC FX (Becton Dickinson Diagnostic Systems, Spark, MD, USA) BC system for 5 days after inoculation with 5–10 mL (per bottle) of the blood drawn from the patients. Conventional ID and ASTs were performed using the MicroScan system (Beckman Coulter, Brea, CA, USA) or Vitek® 2 system (bioMérieux).
9
Vancomycin Resistance Identification
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Antimicrobial susceptibility testing was performed with the automated microdilution method Microscan® System (Beckman-Coulter, Brea, CA, USA) using PM33 panels following the manufacturer’s guidelines. Vancomycin and teicoplanin breakpoints were stablished as indicated by the EUCAST (2021) v. 11. The results obtained were confirmed by real-time PCR for the amplification of the vanA and vanB genes [9 (link)]. In addition, the presence of the vancomycin resistance genes was confirmed a second time by the implementation of the commercial Xpert® vanA/vanB cartridges (Cepheid, Sunnyvale, CA, USA).
10
Vancomycin Resistance Evaluation
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Antimicrobial susceptibility testing was performed with the automated microdilution method Microscan ® System (Beckman-Coulter, CA, USA) using PM33 panels following the manufacturer's guidelines. Vancomycin and teicoplanin breakpoints were stablished as indicated by the EUCAST (2021) v. 11. The results obtained were confirmed by real-time PCR for the amplification of the vanA and vanB genes (9) .
Besides, confirmation of the presence of the vancomycin resistance genes was confirmed a second time by the implementation of the commercial Xpert ® vanA/vanB cartridges (Cepheid, CA, USA).
Besides, confirmation of the presence of the vancomycin resistance genes was confirmed a second time by the implementation of the commercial Xpert ® vanA/vanB cartridges (Cepheid, CA, USA).
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