Chloramphenicol

Antimicrobial susceptibility tests were performed using the agar disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2023 ). Antimicrobial substances included chloramphenicol and linezolid (Becton, Dickinson, Heidelberg, Germany). E. faecalis ATCC 29212 was used as a quality control strain. Zone diameters were interpreted according to the CLSI (2023 ). Zone diameter breakpoints were ≤ 12 mm for chloramphenicol and ≤ 20 mm for linezolid, respectively. For bacterial genera where interpretive criteria for susceptibility testing are not available, the CLSI classification was not applied.

The antimicrobial agents were selected among those commonly carried by integrons. Antimicrobial susceptibility testing was performed using disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for each bacterial species (15 , 16 ), for ampicillin (10 µg), chloramphenicol (30 µg), streptomycin (10 µg) (Becton Dickinson, Sparks, Maryland, USA), cotrimoxazole (25 µg), tetracycline (30 µg), ciprofloxacin (5 µg) (Difco Laboratories, Detroit, Michigan, USA), and trimethoprim (Mast Diagnostics Ltd, Bootle, Merseyside, UK) (5 µg) (15 , 16 ). The antimicrobials were classified as aminoglycosides (STR, AMP), tetracyclines (TE), amphenicols (CHL), fluoroquinolones (CIP), trimethoprim alone (TMP), or in combination with sulfonamides (SXT).

The Kirby–Bauer disc diffusion test was performed using Mueller–Hinton agar plates (Becton, Dickenson and Company) according to Clinical and Laboratory Standard Institute standards [23] using the following antimicrobials: ampicillin (10 µg), cefazolin (30 µg), kanamycin (30 µg), streptomycin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), fosfomycin (50 µg), colistin (10 µg), sulfamethizole (250 µg), and nalidixic acid (30 µg) (Becton, Dickinson and Company).

In total, 214 samples from 138 CF patients (one to four samples per patient) attending the
University Hospital Essen and the Clinic of the Ruhr, West German Lung Centre, Essen, Germany, in
2012 were tested for the presence of DNA from Rasamsonia species as mentioned
above. For culturing, 100 μL of the sputasol-pretreated respiratory samples were
inoculated on malt agar (Oxoid) and incubated aerobically at 36°C for 2 days and then
at 22°C for an additional 8 days.
In addition, 20 sputum samples from 15 CF patients followed up in Angers University Hospital,
Angers, France, were also analysed. Mycological examination of these samples performed in parallel
to the PCR assay consisted of the inoculation of 10-μL aliquots of the digested samples on
CHROMagar Candida (Becton-Dickinson, Franklin, NJ, USA), yeast extract-peptone-dextrose agar (YPDA)
supplemented with chloramphenicol and gentamicin (Becton-Dickinson), Dichloran-rose
bengale-chloramphenicol agar (DRBC), YPDA supplemented with chloramphenicol and cycloheximide, and
erythritol agar. Incubation was carried out at 37°C for 2 weeks, except for the last
two culture media, which were incubated at 25°C. Mould isolates were identified by cultural
and microscopic morphological characteristics.

Antimicrobial susceptibility testing (AST) was conducted on the isolates using the disk-diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [11 ]. Antimicrobial substances included ampicillin, amoxicillin/clavulanic acid, cefazolin, cefotaxime, cefepime, nalidixic acid, ciprofloxacin, sulfamethoxazole-trimethoprim, fosfomycin, azithromycin, nitrofurantoin, streptomycin, kanamycin, gentamicin, chloramphenicol, and tetracycline (Becton, Dickinson, Heidelberg, Germany). Results were interpreted according to CLSI breakpoints for human clinical isolates [11 ]. Multidrug resistance (MDR) was defined as resistance to three or more classes of antimicrobials, counting beta-lactams as one class.

Samples were first plated on Chromocult agar, and then presumptive E. coli colonies were transferred onto MacConkey lactose (MKL) agar for confirmation before replating on Chromocult was performed to ensure pure isolates. We selected up to four E. coli colonies from soil and fecal samples and two from water and surface samples to test for AR. More isolates from soil samples than from water or surfaces were used because we expected higher microbial diversity in soil. Antibiotic sensitivity was assessed using the Kirby-Bauer disc diffusion method (43 (link)) and 12 antibiotics: ampicillin, amoxicillin/clavulanate, cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, enrofloxacin, gentamicin, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline (Becton, Dickinson, Franklin Lakes, NJ). Zones of inhibition were measured after a 24-h incubation period using digital calipers.