Specimens were sent from different healthcare facilities across the country. The specimens obtained encompassed various types of specimens, including urine, blood, wound, eye, body fluids, and other miscellaneous specimens. Only those samples that met the laboratory’s acceptance criteria were accepted and then inoculated into suitable culture media, after which they were incubated at the proper temperature and duration. Staphylococcus aureus and Enterococcus species were identified using one of the three methods: VITEK® 2 Compact (bioMérieux, France), BD phoenix M50 (Becton, Dickinson, USA), or conventional biochemical tests. Conventional biochemical tests involved macroscopic colony characterization (color, size, shape, texture, hemolysis), Gram staining, and biochemical tests (catalase test, coagulase test, L-Pyrrolidonyl Arylamidase (PYR), bile esculin agar, 6.5% sodium chloride tolerance test).
Phoenix m50
Manufactured by BD
Sourced in United States, France
The BD Phoenix M50 is an automated microbiology system designed for the identification and antimicrobial susceptibility testing of microorganisms. It utilizes a compact and efficient design to provide rapid and reliable results for clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Vitek 2 compact, by bioMérieux (3 mentions)
Heratherm igs60, by Thermo Fisher Scientific (2 mentions)
Macconkey agar, by Liofilchem (2 mentions)
Phoenixspec nephelometer, by BD (1 mentions)
Nmic id panel, by BD (1 mentions)
Mha plate, by HiMedia (1 mentions)
Mueller hinton agar (mha), by Liofilchem (1 mentions)
Bactec fx, by BD (1 mentions)
Bact alert virtuo, by bioMérieux (1 mentions)
Maldi biotyper system, by Bruker (1 mentions)
Maldi biotyper smart system, by Bruker (1 mentions)
Bbl trypticase soy agar, by BD (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Vitek 2, by BD (1 mentions)
Clindamycin, by Thermo Fisher Scientific (1 mentions)
Erythromycin, by Thermo Fisher Scientific (1 mentions)
28 protocols using phoenix m50
1
Identification of Staphylococcus and Enterococcus
2
Antimicrobial Susceptibility of S. aureus
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Antimicrobial susceptibility of the suspected S. aureus strains was achieved using a BD Phoenix™ M50 instrument (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) as previously reported [28 (link)]. Susceptibility of the identified S. aureus was tested toward several antibiotics including ampicillin, amoxicillin–clavulanate, cefuroxime, cefoxitin, clindamycin, ciprofloxacin, daptomycin, erythromycin, fusidic acid, gentamicin, imipenem, mupirocin, linezolid, penicillin G, trimethoprim-sulfamethoxazole, nitrofurantoin, tetracycline, teicoplanin, vancomycin, rifampicin, moxifloxacin, and oxacillin. The results of the drug-susceptibility tests were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines document M100S-26 [29 ]. Two indices were used to interpret the obtained results, including the multiple antibiotic resistance index and the antibiotic resistance index as previously described by Abdulhakeem and colleagues [30 (link)].
3
Bacterial Identification and Antibiotic Sensitivity
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Bacterial identification and antibiotic sensitivity testing were performed using a BD Phoenix™ M50 instrument (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) [19 (link)]. Susceptibility of the identified Gram-negative bacteria was performed against a panel of antibiotics: amikacin, ampicillin, aztreonam, cefepime, ceftazidime, cefoxitin, colistin, ceftriaxone, cefuroxime, ciprofloxacin, ertapenem, gentamicin, imipenem, meropenem, nitrofurantoin, piperacillin, tigecycline, trimethoprim/sulfamethoxazole and piperacillin-tazobactam. Identified Gram-positive bacteria were tested against ampicillin, cephalothin, clindamycin, levofloxacin, linezolid, moxifloxacin, nitrofurantoin, penicillin, tigecycline, trimethoprim/sulfamethoxazole and vancomycin. The results of susceptibility testing were reported as ‘susceptible’ or ‘resistant’, according to Clinical and Laboratory Standards Institute guidelines CLSI document M100S-26 [20 ]. Quality control for several bacterial strains, including Acinetobacter baumannii, E. faecalis, K. pneumoniae and E. coli was required to ensure the validity of the minimum inhibitory concentration (MIC) of each antibiotic [21 (link)]. Susceptibility data were presented as percentage of resistant isolates to the total number of isolates recovered from each site or specimen for individual bacterial species.
4
Identifying Acinetobacter and P. aeruginosa
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Specimens that met the laboratory’s acceptance criteria were received and inoculated onto appropriate culture media and incubated at the appropriate temperature and time.26 (link) The identification of Acinetobacter species and P. aeruginosa was achieved using one of the following methods available at the time of testing: Vitek 2 compact (BioMerieux, USA), BD phoenix M50 (Becton, Dickinson, USA), and conventional biochemical tests. For identification using conventional biochemical tests, a macroscopic colony characterization (color, size, shape, and texture) supported with gram staining followed by biochemical tests (Triple sugar iron agar, lysine iron agar, sulfide indole motility, Simmons Citrate Agar, urea agar, and oxidase) was used.26 (link) The biochemical and culture media used by the laboratory from 2017 to 2021 were from the following manufacturers: (Oxoid Ltd., Basingstoke, Hampshire, England), (Liofilchem, Roseto degli Abruzzi, Italy), (Hardy diagnostics, Santa Maria, California, and Springboro, Ohio, United States), (Biomark, Dalviwadi, Dhairi Pune, Maharashtra, India), (Accumixx, Verna, Goa, India), and (HIMEDIA, Mumbai, Maharashtra, India).
5
Automated Bacterial Identification and Antibiotic Susceptibility
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Blood agar and MacConkey agar plates were inoculated with blood culture broths from the flagged positive blood culture bottles showing Gram-negative rods and incubated in a 5% CO2-rich environment at 37°C for 18–24 hours. Standardized bacterial inoculum (0.5 McFarland) was prepared from bacterial colonies isolated as pure culture on the solid media with the help of BD PhoenixSpec nephelometer (Becton Dickinson, USA). NMIC/ID panel (Becton Dickinson, USA) was inoculated with the standard bacterial suspension per the manufacturer’s instructions, and the panel was loaded into BD Phoenix M50 (Becton Dickinson, USA) for automated ID and AST of the bacterial isolate.
The Kirby-Bauer disk diffusion method was also performed using 10-cm diameter Mueller Hinton agar (MHA) plates (HiMedia Laboratories, India) and different antibiotic disks (HiMedia Laboratories, India) with the same standard bacterial inoculum and incubated at 35°C ± 2°C in ambient air for 16–18 hours. AST results were interpreted as per the performance standards for AST by CLSI (12 ).
The Kirby-Bauer disk diffusion method was also performed using 10-cm diameter Mueller Hinton agar (MHA) plates (HiMedia Laboratories, India) and different antibiotic disks (HiMedia Laboratories, India) with the same standard bacterial inoculum and incubated at 35°C ± 2°C in ambient air for 16–18 hours. AST results were interpreted as per the performance standards for AST by CLSI (12 ).
6
Bacterial Identification and Antibiotic Resistance
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In all patients, a tissue specimen was incubated in thioglycolate broth via needle aspiration, and then the samples were sent to the Microbiology Center of Smart Health Tower (Sulaymaniyah, Iraq). Four types of agar were used in the culturing process: blood agar base, chocolate agar (cat. no. 610005; Liofilchem S.r.l.), MacConkey agar (cat. no. 610028; Liofilchem S.r.l.), and Mueller-Hinton agar (cat. no. NCM0036A; Neogen). The culturing was conducted by the streak plate method with an overnight incubation at 37˚C (Heratherm IGS60; Thermo Fisher Scientific, Inc.). The bacterial identity, antibiotic sensitivity, and resistance were determined by BD Phoenix™ M50 (Becton, Dickinson and Company). A manual technique, disc diffusion by the Kirby-Bauer method, was also performed to detect antibiotic sensitivity and resistance, and Clinical and Laboratory Standards Institute (CLSI) Standards: Guidelines for Health Care Excellence (CLSI, USA; https://clsi.org/standards/products/microbiology/documents/m100/ ) was used for interpretation. All of the procedures were performed according to the manufacturer's instructions.
7
Bacterial Strain Characterization and Storage
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S. aquimarina CIP 108633T was obtained from the Pasteur Institute Collection (CIP), stocked in 20% (v/v) glycerol at −80 °C and routinely propagated on TYP agar at 20 °C.
A panel of both Gram-positive and Gram-negative bacterial pathogens, stored at −80 °C and routinely propagated on Mueller Hinton agar, were utilized as follows: Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 13360, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25992, Klebsiella pneumoniae ATCC 10031, Pseudomonas aeruginosa O1, and Salmonella enterica serovar Typhimurium ATCC 14028. Five clinical isolates of S. aureus were also utilized. These strains were collected and characterized at the Laboratory of Microbiology of the “Luigi Vanvitelli” University Hospital using a BD Phoenix M50 instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Their origin and drug resistance are reported in the following table (Table 1 ) [31 (link)].
A panel of both Gram-positive and Gram-negative bacterial pathogens, stored at −80 °C and routinely propagated on Mueller Hinton agar, were utilized as follows: Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 13360, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25992, Klebsiella pneumoniae ATCC 10031, Pseudomonas aeruginosa O1, and Salmonella enterica serovar Typhimurium ATCC 14028. Five clinical isolates of S. aureus were also utilized. These strains were collected and characterized at the Laboratory of Microbiology of the “Luigi Vanvitelli” University Hospital using a BD Phoenix M50 instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Their origin and drug resistance are reported in the following table (
8
Antimicrobial Susceptibility and ESBL Detection
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An antimicrobial susceptibility testing and ESBL-type enzymes synthesis were determined using BD Phoenix™ M50 instrument (Becton-Dickinson, Franklin Lakes, NJ, USA) with NMIC-402 panels, both applied according to the manufacturer’s instructions.
9
Antimicrobial Susceptibility Testing Protocols
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The disk diffusion methods used E. coli ATCC 25922 and P. aeruginosa ATCC 27853 strains for QC. E. coli ATCC 25922, P. aeruginosa ATCC 27853, E. coli ATCC 35218, and Klebsiella pneumoniae ATCC 700603 strains were used for QC in the AST by BD Phoenix M50 (Becton Dickinson, USA) system.
10
Standardized Microbial Culture Protocols
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For blood culture, two to four bottles with 8–10 mL of blood per bottle were routinely obtained and inoculated into aerobic and anaerobic blood culture bottles, which were subsequently incubated at 35 ± 2°C in BACT/ALERT VIRTUO (bioMérieux, France) or BD BACTEC FX (Becton Dickenson, USA) automated analyser for up to five days. Subculture was performed on fresh sheep blood, MacConkey, and chocolate agars when the machine indicated a positive signal. Organisms were identified using a BD Phoenix M50 (Becton Dickenson, USA) or Vitek 2 Compact (bioMérieux, France) automated identification and AST testing systems. For sputum culture, sample quality was assessed using Bartlett's grading system [27] (link), followed by plating onto selective media for bacterial isolation. For bronchoalveolar lavage aspirate (BAL) and urine culture, samples were quantitatively plated onto selective media, and bacterial identification and AST were performed for known pathogens from BAL with a colony count ≥104 cfu/mL and uropathogens with a colony count ≥105 cfu/mL. When multiple A. baumannii organisms were isolated from the same patient, only the first isolate was included for analyses.
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