Bacterial strains
Reference strains (n = 16) with resistance or susceptibility to the analyzed antibiotics cefotaxime, meropenem, and ciprofloxacin were obtained from the Culture Collection University of Gothenburg (CCUG), Sweden; American Type Culture Collection (ATCC); and Lund University Hospital, Dept. of Clinical Microbiology (CMRS), Sweden (Table 1). Patient isolates (n = 45) were obtained from Dep. of Clinical Microbiology, Region Skåne, Lund Sweden. All patient isolates belonged to either Escherichia coli (n = 38), Klebsiella. pneumoniae (n = 6), Enterobacter cloacae (n = 1) or Citrobacter freundii (n = 1).
Table 1
Resistance pattern of the different control strains used in the study.
Source | Number | Species | Resistance genes | MIC by Etest |
CTX (mg/L) | MER (mg/L) | CIP (mg/L) |
CCUG | 10785 | K. pneumoniae | --- | 0.015 | 0.015 | 0.002 |
CCUG | 8619400 | E. coli | --- | 0.06 | 0.03 | 0.015 |
CCUG | 17620 | E. coli | --- | 0.094 | 0.012 | 0.015 |
CCUG | 58538 | E. coli | MOX | 128 | 0.06 | 0.03 |
CCUG | 58543 | E. coli | CMY-2 | 64 | 0.03 | 0.06 |
CCUG | 58547 | K. pneumoniae | VIM | > 256 | > 32 | > 32 |
CCUG | 59351 | E. coli | CTX-M 15 | > 256 | 0.015 | > 32 |
CCUG | 59357 | E. coli | SHV12/5A | 3 | 0.008 | 0.03 |
CCUG | 59360 | K. pneumoniae | SHV12/5A | 2 | 0.015 | 0.03 |
CMRS | 756 | E. coli | SHV | 2 | 0.008 | 0.015 |
CMRS | 503392 | K. pneumoniae | OXA-48 | 0,5 | 0.19 | 0.023 |
CMRS | 101076 | K. pneumoniae | NDM, CTX-M1(15) | 32 | 4 | > 32 |
CMRS | 549078 | E. coli | DHA | 2 | 0.015 | 0.12 |
CMRS | 518178 | K. pneumoniae | DHA, CTX-M1(15) | > 256 | 0.015 | 0.5 |
CMRS | 101067 | K. pneumoniae | KPC | > 256 | > 32 | > 32 |
CMRS | 500182 | K. pneumoniae | KPC, CTX-M1(15), CMY-2 | > 256 | > 32 | > 32 |
Environmental bacterial strains (n = 183) were isolated from water samples from Helge Å river, Kristianstad, Sweden. All environmental isolates were identified to species level by MALDI-TOF MS (Bruker, Germany) and stored in Brain Heart Infusion Broth (Sigma Aldrich, USA) with 15% glycerol at -25°C and cultured on Müller Hinton agar plates (Sigma Aldrich, USA) before use in the assay. The strains used were identified as belonging to the following species: E. coli (n = 104), K. pneumoniae (n = 47), Ochrobactrum intermedium (n = 8), Acinetobacter baumannii (n = 9), Acinetobacter calcoaceticus (n = 4), Kluyvera ascorbata (n = 4), E. cloacae (n = 4) and one each of C. freundii, Raultella ornithinolytica, and Serratia fonticola.
Phenotypical identification of ESBL
All environmental isolates were originally isolated on ESBL agar plates (ESBL ChromAgar, Biomérieux, France) and phenotypically verified as ESBL by the D68C Mast® test (Mast Diagnostica, Germany). The resistance was identified using disc diffusion or E-test for cefotaxime, meropenem and ciprofloxacin (Biomérieux, France). The antibiotic resistance of the clinical samples was identified according to the standard routine diagnostic protocols at Department of Clinical Microbiology, Region Skåne, using disc diffusion or E-test.
Bacterial preparation
The environmental strains were cultured on Müller Hinton agar before the analysis. Clinical strains were obtained from positive blood cultures at the clinic. Reference strains were inoculated blood culture bottles (BD Bactec Plus Aerobic/F Culture and BD Bactec Plus Anaerobic Lytic/F) and incubated in an automated BACTEC FXTM blood culture system (Becton Dickinson, Franklin Lakes, NJ) until flagged as positive. The preparation of the bacteria was as described in Axelsson et al., (2019) (9). Briefly, 1 ml blood from the aerobic bottles were added to 200 µl Saponin (5%) and 1 ml of the mixture was then added to 200 µl lysis buffer (MALDI Sepsityper KIT, Bruker, Bremen, Germany). The preparation from the anaerobic bottles were treated the same except that no saponin was added. The samples were subsequently centrifuged at 13 000 x g for two minutes and the supernatant removed. The pellet was resuspended in 1 ml sterile MQ water. After an additional centrifugation at 13 000 x g for two minutes, the supernatant was removed and the pellet resuspended in Müller Hinton Cation Adjusted Broth (MHCA, Sigma-Aldrich, USA) to McFarland 1,0 before the analysis.
Incubation time and antibiotic concentration
Incubation time for the bacterial strains in the assay, with and without antibiotics, was set to 90 minutes, based on previous findings (9). For titration of break point concentrations of the antibiotics, fresh bacterial isolates (CCUG 58543; 10785, and CMRS 101067) were incubated in MHCA broth at 37°C for 90 minutes with agitation with concentrations of the antibiotics ranging from 0 to 128 mg/L. All samples were at least performed in duplicates.
Assay performance
The microplates were prepared with 10 samples and positive and negative controls with and without 3 different antibiotics in duplicates. The absorbance was measured at 600 nm in the beginning of the assay (time 0 minutes) and after 90 minutes of incubation at 37°C with agitation in the plate reader Infinite 200 Pro (Tecan, Austria). The data from the reader were transferred to Excel where the data evaluation was performed.
Data evaluation
First, in order to approve the test, the bacterial culture without added antibiotics had to grow above a threshold after 90 minutes incubation. The growth threshold was set to require an increase in absorbance of more than 50%. Second, the absorbance value of each suspension after 90 minutes incubation (X90) with added antibiotic was compared to the start (X0) absorbance in the same well, and the susceptibility/resistance was calculated according to:
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Susceptible:\(\frac{{X}_{90}}{\text{1,5}}\le {X}_{0}\)
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Resistant:\(\frac{{X}_{90}}{\text{1,5}}>{X}_{0}\)
The obtained results (susceptible/resistant) were compared to the susceptibility tests (E-test or disc diffusion tests as described above) used for routine diagnostics. Differences between the assay and the routine test were expressed as major error (false resistance) and very major error (false susceptibility). Sensitivity was defined as the number of true resistant isolates over the total number of resistant isolates. Specificity was defined as the number of true susceptible isolates over the total numbers of susceptible isolates. Accuracy was defined as the number of true resistant and true susceptible isolates over the total number of replicates.