Eleven K. pneumoniae strains were used in this study including four carbapenem-resistant strains, the susceptible isolate ATCC 13883, and six other isolates from a panel of strains maintained by our laboratory consisting of both carbapenem-resistant and susceptible strains (Table 1 ). In this study, the term ‘carbapenem-resistant’ implies that the strain was resistant to at least one of the carbapenems, namely ertapenem, meropenem, or imipenem. The isolates were identified using a Bruker MALDI Biotyper CA System (Bruker Daltonik GmbH).
Maldi biotyper ca system
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The MALDI Biotyper CA System is a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instrument designed for the rapid identification of microorganisms. The system utilizes protein fingerprinting to provide fast and accurate identification of a wide range of microorganisms, including bacteria, yeasts, and fungi.
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8 protocols using maldi biotyper ca system
1
Characterizing Carbapenem-Resistant K. pneumoniae
2
Microbial Identification in NSTI
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After debridement, NSTI samples were sent to the Clinical Microbiology Laboratory at the University Medical Center, Lubbock, Texas, as per standard of care. The samples were both gram stained and initially grown on applicable growth medium under aerobic and anaerobic conditions, per Clinical Laboratory Standards Institute (CLSI) guidelines. Bacteria were identified by mass spectrometry using the Bruker MALDI Biotyper CA System, a Matrix‐Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI‐TOF MS) identification system.
3
Bile Culture Isolates in Biliary Cancer
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The bile samples were inoculated for aerobic/facultative-anaerobic bacteria isolation on agar blood+ blood sheep 5%, McConkey agar, and Salmonella-Shigella agar media (Becton-Dickson). After 24h of incubation in aerobic conditions, the growth colonies were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (MALDI Biotyper CA System, Bruker Daltonics Inc., USA) as previously published [43 (link)]. Microbiological records were systematically checked for the corresponding culture reports. Demographic data and diagnosis were collected from the patient’s electronic medical history.
The epidemiological and clinical characteristics of the surgical setting were the same as previously reported [20 (link)]. This study was conducted among hospitalized adult patients using the International Classification of Diseases (ICD-10). The hospital setting is the Policlinic University Hospital of Palermo, Italy. After ERCP, the enrolled patients were referred to a specialist cancer center.
To compare the isolates from the bile culture of patients with biliary cancer versus patients with pancreatic cancer, we stratified the patients into two groups, i.e., patients with extra-pancreatic biliary tract carcinoma (EPC) and pancreatic cancer (PC).
The epidemiological and clinical characteristics of the surgical setting were the same as previously reported [20 (link)]. This study was conducted among hospitalized adult patients using the International Classification of Diseases (ICD-10). The hospital setting is the Policlinic University Hospital of Palermo, Italy. After ERCP, the enrolled patients were referred to a specialist cancer center.
To compare the isolates from the bile culture of patients with biliary cancer versus patients with pancreatic cancer, we stratified the patients into two groups, i.e., patients with extra-pancreatic biliary tract carcinoma (EPC) and pancreatic cancer (PC).
4
Microbial Identification and Antibiotic Susceptibility
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Joint fluid, biopsies and prosthetic components were cultured according to [42 (link)] with an incubation period of 14 days (see Additional file 7 ). Species identification was done with a MALDI Biotyper CA System (Bruker Daltonics, Germany). Antimicrobial susceptibility testing was carried out as above [41 ]. The S. aureus isolate from prosthetic components was designated SAU060112.
5
Clostridium striatum infections in CCO patients
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A total of seven patients with CCO were recognized and retrospectively reviewed, and C. striatum was isolated from intraoperative (deep tissues/bone) cultures in a polymicrobial setting. The specimens were inoculated on media (Blood, Chocolate, MacConkey, Columbia Nalidixic acid, Thioglycollate broth) and incubated for 7 days before finalization. All cultures were initially reported as diphtheroids by our institutional clinical microbiology laboratory. Diphtheroids were further identified per request of the Infectious Disease service when a pathogenic role was suspected, by using RapID™ CB PLUS System and verified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI TOF MS, Bruker MALDI Biotyper® CA System, reference library version 2021 claim 6). Antibiotic susceptibility testing could only be performed on the last patient in this series, once susceptibility to this organism became available at our institution through a send-out to ARUP Laboratories (Salt Lake City, Utah, USA) by broth microdilution. Cure was defined as granulation of the infected wound and resolution of clinical symptoms at outpatient follow-up between 4 and 8 weeks.
6
Antimicrobial Susceptibility Profiling of E. coli
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Swabs were inoculated on MacConkey agar and incubated at 35 ± 1 °C for 24 h, and lactose-fermenting colonies were identified by MALDI-TOF (Bruker MALDI Biotyper CA System) [20 (link)]. E. coli isolates were then inoculated on two MacConkey agar plates, one supplemented with ceftriaxone (2 μg/mL) and the other with ciprofloxacin (2 μg/mL). Plates were incubated at 35 ± 1 °C for 24 h.Antimicrobial susceptibility tests were performed by disc diffusion method, according to guidelines [21 ]. The following antibiotics were tested: amikacin, amoxicillin/clavulanate, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, cotrimoxazole, ertapenem, fosfomycin, gentamycin, imipenem, meropenem and tetracycline. Minimum inhibitory concentrations (MICs) for three fluoroquinolones (ciprofloxacin, enrofloxacin and levofloxacin) were determined for strains that presented ciprofloxacin resistance by the disc diffusion method. Interpretation of both disc diffusion and MIC results were made according to CLSI guidelines [22 ,21 ]. Extended-spectrum beta-lactamases (ESBL) production was observed through double disk synergy test, using amoxicillin/clavulanate, ceftriaxone, ceftazidime, cefotaxime and cefepime.
7
Genome Surveillance of Multidrug-Resistant Bacteria
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All bacterial isolates were recovered from patients, except one from environment surveillance (M170981), at the Clinical Microbiology Laboratory of Westchester Medical Center (WMC), a tertiary-care hospital in suburban New York City. Along with culture and identification by routine laboratory tests, these bacterial isolates were verified using the MALDI Biotyper CA system (Bruker, Billerica, MA, USA) and subjected to the MicroScan WalkAway automated system (Beckman Coulter, Brea, CA, USA) for antimicrobial susceptibility tests. During our WGS clinical surveillance of multidrug-resistant bacteria from November 2016 to the end of 2018, a total of 147 Acinetobacter sp. isolates were collected from 34 patients and one environmental swab. A local A. baumannii clinical isolate PB364 was collected in 2016 from a patient too and subjected to whole genome assembly with both short- and long-read NGS—the genome of which was used as a reference in data analysis. Five vancomycin-resistant Enterococcus sp. clinical isolates were collected in February 2019. When hospital-associated VRE transmission was suspected, they were compared to two E. gallinarum clinical isolates also collected in February 2019 and four Enterococcus sp. clinical isolates in stock collected previously, from 2013 to 2018.
8
Identifying Acinetobacter Species and Antibiotic Resistance
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Identification of genomic species of Acinetobacter was performed by matrix-assisted laser desorption/ionization time-offlight mass spectrometry, MALDI-TOF MS (MALDI Biotyper CA system; Bruker Daltonics, Madrid, Spain). Detection of bla OXA-51 was performed by PCR, 7 (link) whole genome sequencing (WGS) and the SpeciesFinder 1.2 tool (https://cge.cbs.dtu.dk/services/ SpeciesFinder/). Antimicrobial susceptibility testing was performed with the Microscan Neg MIC Panel, Type 44 (Beckman Coulter, Inc, Madrid, Spain). Imipenem and meropenem susceptibility testing was performed by Etest (LioChem Inc, Madrid, Spain) and disk diffusion (Oxoid, Madrid, Spain) in Mueller-Hinton agar plates (Oxoid). Susceptibility to colistin was tested by microdilution using the UMIC kit (Biocentric; Bandol, France).
The isolate was genotyped by PFGE using the restriction enzyme ApaI, 8 multilocus sequence typing (MLST) was performed according to the Institut Pasteur scheme (https://pubmlst. org/abaumannii/) and using WGS (see below) and the MLST 1.8 tool (https://cge.cbs.dtu.dk/services/MLST/).
The isolate was genotyped by PFGE using the restriction enzyme ApaI, 8 multilocus sequence typing (MLST) was performed according to the Institut Pasteur scheme (https://pubmlst. org/abaumannii/) and using WGS (see below) and the MLST 1.8 tool (https://cge.cbs.dtu.dk/services/MLST/).
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