The A. baumannii AB5075-UW strain (NR-49900) was obtained from the Biodefense and Emerging Infections Research Resources Repository for distribution by BEI Resources, the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), while strain ATCC 17978 was a kind gift from P. Visca. The routine growth and plating were carried out in Luria-Bertani broth (LB) and 1.5% agar plates (Difco, Milan, Italy). The antibiotic susceptibility profiling was performed with the VITEK 2 system (bioMérieux, Florence, Italy). Imipenem (IMP) was purchased from Alfa Aesar (Italy). The MIC values were determined by MICROSCAN WalkAway (Siemens, Erlangen, Germany). To determine the appropriate imipenem sub-MIC values granting comparable growth curves, different concentrations of IMP (ranging from 1 to 8 µg/mL) were tested in a broth dilution assay. The growth kinetics were monitored by measuring the optical density (OD) at 600 nm and viable counts every 30 min (initial inoculum: 1 × 106 colony-forming units per mL, CFU/mL) over a period of 8 h.
Microscan walkaway
Manufactured by Siemens
Sourced in Germany, United States, Japan
The MicroScan WalkAway is a fully automated microbiology system designed for the identification and antimicrobial susceptibility testing of a wide range of microorganisms. The system provides accurate and reliable results to support clinical decision-making.
Automatically generated - may contain errors
Lab products found in correlation
Bactec 9240 system, by BD (6 mentions)
Vitek 2, by bioMérieux (5 mentions)
Phoenix system, by BD (5 mentions)
Bactec fx system, by BD (5 mentions)
Etest, by bioMérieux (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
M 1 c e strips, by Thermo Fisher Scientific (1 mentions)
Etest strips, by bioMérieux (1 mentions)
Maldi biotyper system, by Bruker (1 mentions)
Abi prism 3130 sequencer, by Thermo Fisher Scientific (1 mentions)
30 protocols using microscan walkaway
1
Antibiotic Susceptibility of A. baumannii
2
Acinetobacter Isolation and Identification
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Acinetobacter spp. that were cultured from sterile body fluids of children underwent species identification and antimicrobial susceptibility testing using the following automated systems: from 2001, the MicroScan Walk-Away (Siemens Healthcare Diagnostics, Deerfield, IL, USA), from 2011, the Vitek-2 (bioMerieux, Marcy L’Etoile, France), and from August 2020, an additional M50 (BD Diagnostic Systems, Sparks, MD, USA). These isolates were collected and stored in inositol stocks at -70 °C. Collected isolates were thawed and cultured on MacConkey agar plates overnight in incubators set at 37 °C. The colonies were then collected and underwent chromosomal DNA extraction using a DNA extraction kit (DNeasy Kit; Qiagen GmbH) according to the instructions given by the manufacturer. The extracted DNA were evaluated by NanoDrop (Thermo Fisher Scientific, Inc., Waltham, MA, US) and then stored at -70 °C until use.
3
Carbapenem-Resistant Enterobacter Isolates
4
Antibiotic Resistance Profiling of S. aureus Isolates
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Isolates from children with SSTI and bone and joint infection due to S. aureus were collected during the study period and stored at −80°C until analysis. Isolates related to outbreaks were excluded. Isolation of S. aureus and antimicrobial susceptibility tests were performed in the clinical microbiology laboratory using an automatic system (MicroScan Walk-Away; Siemens Healthcare Diagnostics, Deerfield, MA, USA). Antimicrobial susceptibility testing data of 9 antimicrobial agents were obtained: oxacillin, penicillin, gentamicin, ciprofloxacin, clindamycin, erythromycin, rifampicin, trimethoprim/sulfamethoxazole, and vancomycin. Confirmatory tests for MRSA isolates were done by mecA gene polymerase chain reaction (PCR) during SCCmec typing.15 (link) Resistance to antibiotics for each isolate was defined according to the Clinical and Laboratory Standards Institute guidelines published in 2019.
5
Antimicrobial Resistance Patterns of MRSA
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MRSA strains from 1107 samples (blood, sputum, skin, wound, bile, and pharynx) from outpatients were collected at Showa University Hospital in Japan between September 2009 and March 2017. The hospital is located in the Southern part of Tokyo. It has 1000 beds and an average of 44,500 to 53,000 outpatients per month.
Identification and antimicrobial susceptibility tests were performed by Microscan WalkAway using the Pos Comb 3.1J panel (Siemens Healthcare Diagnostics. Deerfield, IL). Susceptibility intermediate and resistant (SIR) categories of penicillin G, oxacillin, ampicillin, cefazolin, cefotiam, imipenem, gentamicin, erythromycin, clindamycin, minocycline, vancomycin, levofloxacin, teicoplanin, and linezolid were classified according to the Clinical Laboratory Standards Institute guidelines (CLSI, 2008 ).
Identification and antimicrobial susceptibility tests were performed by Microscan WalkAway using the Pos Comb 3.1J panel (Siemens Healthcare Diagnostics. Deerfield, IL). Susceptibility intermediate and resistant (SIR) categories of penicillin G, oxacillin, ampicillin, cefazolin, cefotiam, imipenem, gentamicin, erythromycin, clindamycin, minocycline, vancomycin, levofloxacin, teicoplanin, and linezolid were classified according to the Clinical Laboratory Standards Institute guidelines (CLSI, 2008 ).
6
Synergistic Effect of Enzyme Enhancers
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The MIC represents the concentration of antibiotics required to inhibit the growth of a planktonic bacterial population, which can reflect the auxiliary effect of enzymes. For P. aeruginosa wild-type strain PAO1, the MIC was estimated using the microdilution method. In a 96-well microtiter plate, the concentration of the antibiotics piperacillin and amikacin ranged from 0.5 to 512 μg/mL. The experiments were divided similarly into four groups. Separately, 100 μg/mL AidHA147G, 25 μg/mL PslG, and the mixed enzymes (100 μg/mL AidHA147G and 25 μg/mL PslG) were added to the three experimental groups. For the control group, we added the same volume of inactivated enzyme liquid. The 96-well plates were incubated for 12 h at 37°C. The antibacterial activities of antibiotics were tested spectrophotometrically at 600 nm using a 96-well plate and a multiscanner.
For the clinical isolates, MicroScan MIC panels (MicroScan WalkAway; Siemens, Germany) were chosen to evaluate drug resistance. The experiments were divided similarly into four groups. Separately, 100 μg/mL AidHA147G, 25 μg/mL PslG, and the mixed enzymes (100 μg/mL AidHA147G and 25 μg/mL PslG) were added to the three experimental groups. For the control group, we added the same volume of inactivated enzyme liquid. In addition, the main methods of operation were performed according to the manufacturer’s instructions.
For the clinical isolates, MicroScan MIC panels (MicroScan WalkAway; Siemens, Germany) were chosen to evaluate drug resistance. The experiments were divided similarly into four groups. Separately, 100 μg/mL AidHA147G, 25 μg/mL PslG, and the mixed enzymes (100 μg/mL AidHA147G and 25 μg/mL PslG) were added to the three experimental groups. For the control group, we added the same volume of inactivated enzyme liquid. In addition, the main methods of operation were performed according to the manufacturer’s instructions.
7
In vitro Antimicrobial Susceptibility Testing
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In vitro antimicrobial susceptibility testing was performed by MicroScan WalkAway (Siemens) microdilution system following the Clinical and Laboratory Standards Institute (CLSI) guidelines [9 ]. Thirteen antimicrobial agents were tested, including (range of tested Minimal Inhibitory Concentration in μg/mL, MIC): ticarcillin (8-32), piperacillin-tazobactam (8/4-64/4), aztreonam (1-16), cefepime (1-16), ceftazidime (1-32), meropenem (1-8), imipenem (1-8), tobramycin (2-8), gentamicin (1-8), amikacin (8-32), levofloxacin (1-4), ciprofloxacin (0.5-2), and colistin (2-4). Extended-spectrum-beta-lactamase (ESBL), metallo-beta-lactamase (MBL), and class A carbapenemase phenotypes were determined by double-disc synergy tests [10 (link)–12 (link)].
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In vitro antimicrobial susceptibility testing was performed by MicroScan WalkAway (Siemens) microdilution system following the Clinical and Laboratory Standards Institute (CLSI) guidelines [9 ]. Thirteen antimicrobial agents were tested, including (range of tested Minimal Inhibitory Concentration in μg/mL, MIC): ticarcillin (8-32), piperacillin-tazobactam (8/4-64/4), aztreonam (1-16), cefepime (1-16), ceftazidime (1-32), meropenem (1-8), imipenem (1-8), tobramycin (2-8), gentamicin (1-8), amikacin (8-32), levofloxacin (1-4), ciprofloxacin (0.5-2), and colistin (2-4). Extended-spectrum-beta-lactamase (ESBL), metallo-beta-lactamase (MBL), and class A carbapenemase phenotypes were determined by double-disc synergy tests [10 (link)–12 (link)].
8
Evaluating MRSA Vancomycin Susceptibility
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Bacterial isolates: Seventy-five MRSA from Mãe de Deus
hospital, a 400-bed general hospital in Porto Alegre, were evaluated in Southern
Brazil. Methicillin resistance was first characterized by automated system
(MicroScan Walk Away, Siemens®), MRSA phenotype was
confirmed by molecular methods (mecA gene), described
elsewhere20 (link). Isolates were
maintained (-20°C) in 10% Skim Milk (Difco, Detroit,
USA) with 10% glycerol.
Determination of Minimal Inhibitory Concentration: Vancomycin MICs were determined by BM6 and by the following techniques, according to the
manufacturer's instructions: E-test® strips
(BioMérieux, Marcy l'Étoile, France),
M.I.C.E.® strips (Oxoid, Thermo Fisher Scientific,
Basingstoke, UK), MicroScan and commercial panels for MIC detection
(PROBAC®). Besides, the agar dilution screening test
was performed with 3 µg/mL of vancomycin, as proposed by BURNHAM, WEBER
& DUNNE1 (link). A
vancomycin-susceptible strain (ATCC 25923) and a positive control
(Enterococcus faecalis carrying vanA
gene) were used for all methodologies.
Statistical analysis: Descriptive statistics were applied,
and data were evaluated by ANOVA, followed by the Tukey post hoctest. The results were processed using the Statistical Package for Social
Sciences (SPSS) 17.0. Results statistically significant were
considered when p <0.05.
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hospital, a 400-bed general hospital in Porto Alegre, were evaluated in Southern
Brazil. Methicillin resistance was first characterized by automated system
(MicroScan Walk Away, Siemens®), MRSA phenotype was
confirmed by molecular methods (mecA gene), described
elsewhere20 (link). Isolates were
maintained (-20°C) in 10% Skim Milk (Difco, Detroit,
USA) with 10% glycerol.
manufacturer's instructions: E-test® strips
(BioMérieux, Marcy l'Étoile, France),
M.I.C.E.® strips (Oxoid, Thermo Fisher Scientific,
Basingstoke, UK), MicroScan and commercial panels for MIC detection
(PROBAC®). Besides, the agar dilution screening test
was performed with 3 µg/mL of vancomycin, as proposed by BURNHAM, WEBER
& DUNNE1 (link). A
vancomycin-susceptible strain (ATCC 25923) and a positive control
(Enterococcus faecalis carrying vanA
gene) were used for all methodologies.
and data were evaluated by ANOVA, followed by the Tukey post hoctest. The results were processed using the Statistical Package for Social
Sciences (SPSS) 17.0. Results statistically significant were
considered when p <0.05.
9
Bacteremia Isolate Collection and Analysis
10
Methicillin-resistant Staphylococcus aureus Detection
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After collecting the samples, nasal swabs were transported to the laboratory immediately and plated directly onto mannitol salt agar, incubated at 35°C with 5% CO2 for 48 hours, and then maintained at 25°C for 5 days. Colonies that were mannitol-fermenting, catalase-positive, and coagulase-positive were screened for methicillin resistance on Mueller–Hinton agar supplemented with sodium chloride cations and oxacillin at 4 μg/mL according to the Clinical Laboratory Standards Institute guidelines. Antimicrobial susceptibility testing for agents was measured by the broth microdilution method and automated equipment (MicroScan WalkAway, Siemens, Munich, Germany).8 (link)
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