Microscan

Spontaneous sputum samples were collected within 24 hours of presentation of ECOPD symptoms as well as from clinically stable COPD cases. All the sputum samples were collected in sterile containers and transferred to the microbiological laboratory within 2 hours of collection for Gram staining and bacterial culture. All the sputum samples were classified according to Murray-Washington criteria, thus grade IV (10–25 epithelial cells and > 25 leucocytes per field) and grade V (< 10 epithelial cells and > 25 leucocytes per field) were cultured quantitatively and a bacterial load ≥10⁶ colony forming units (CFU)/ml was considered as significant. Culture was performed in non-selective (sheep blood agar, chocolate agar) and selective media (MacConkey agar) at 37°C for 48 hours in 5% CO₂, and bacterial species identification was conducted biochemically (Microscan, Siemens). The identified microorganisms were classified as potentially pathogenic microorganisms (PPM) including Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus methicillin susceptible or resistant, Moraxella catarrhalis, and Gram negative-bacilli, and non-PPM included Candida spp., Streptococcus viridans, Neisseria spp., Staphylococcus epidermidis and Corynebacterium spp.

The comparator method(s) for organism identification were the site’s SOC, including traditional culture, FDA-cleared matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) (i.e., bioMérieux Vitek MS and Bruker Biotyper), and automated phenotypic identification and antibiotic susceptibility platforms (e.g., Becton, Dickinson [BD] Phoenix, bioMérieux Vitek 2, and Siemens MicroScan). The phenotypic methods listed above varied at different sites and served as the site’s reference identification standard. Due to issues with organism misidentification, samples with a member of the Acinetobacter calcoaceticus-baumannii complex (15 (link)) or Candida parapsilosis (16 (link)) identified by SOC were confirmed using analytically validated PCR amplification assays followed by bidirectional sequencing (PCR/sequencing) or 16S sequencing by Laboratory Corporation of America Holdings (LabCorp [Burlington, NC]) according to FDA clinical trial instructions outlined in the 510k summary (10 ). The comparator methods for ARGs were analytically validated by real-time PCR amplification assay(s) followed by bidirectional sequencing, developed and performed by LabCorp.

Minimum inhibitory concentrations (MICs) were determined using custom dehydrated MicroScan broth microdilution (Siemens Medical Solutions Diagnostics), following Clinical and Laboratory Standards Institute (CLSI) guidelines (Cockerill, 2012 ) and relative susceptibility interpretations were based on CLSI clinical breakpoints (CLSI, 2017 ).
Thirteen antimicrobial agents commonly used to treat diarrhea were tested. Results were only included in the analysis when corresponding quality control isolate test results were in accordance with CLSI, 2017 guidelines, which were unchanged between 2014 and 2017 and therefore within an acceptable range.

All samples were routinely cultured on MacConkey and blood agar plates. Blood and sputum were also cultured on chocolate agar. All suspected colonies were identified by gram staining, colony characteristics, motility, and so forth. Strains were identified to the species level with bioMérieux API20E and confirmed by Siemens MicroScan Negative ID panel Type 2. MICs were determined using MicroScan dehydrated broth microdilution panel negative MIC Type 37 (Siemens Medical Solutions Diagnostics, West Sacramento, CA), following the manufacturer's guidelines and Clinical Laboratory Standards Institute (CLSI) [15 ]. MICs were interpreted following CLSI guidelines, including the new clinical breakpoints published in 2010 for carbapenems [16 ]. ESBL detection: phenotypic—the ESBL detection was done as was recommended by the CLSI confirmatory procedure, by using cefotaxime (30 μg) and ceftazidime (30 μg) discs alone and in combination with clavulanic acid discs. K. pneumoniae (ATCC-700603) were used as the controls throughout the study [17 ]. The ESBL production was confirmed by MicroScan MIC 37 panel using combination of cefotaxime/K clavulanate (Cft/CA) and ceftazidime/K clavulanate (Caz/CA) [18 (link)].