Baseline fosfomycin susceptibility and heteroresistance of the representative K. pneumoniae isolates
| Strain # . | Standard susceptibility testing . | Heteroresistance . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| AD MIC (mg/L)a . | DD (inhibition zone, mm)b . | High inoculum AD (mg/L)c . | High inoculum DD (mm)d . | Disc elution (time, h)e . | RSP proportion in CAMHBf . | RSP proportion in SHUf . | |||
| Clinical isolates | |||||||||
| INF014 | 2 | WT | 25 | WT | 64 | 22 | POS (72) | 1E-07 | – |
| INF321 | 2 | WT | 24 | WT | 64 | 22 | POS (48) | 1E-06 | – |
| INF215 | 4 | WT | 24 | WT | 64 | 17 | POS (48) | 1E-07 | – |
| INF174 | 4 | WT | 23 | WT | 128 | 19 | POS (48) | 4E-07 | – |
| INF044 | 8 | WT | 23 | WT | 256 | 19 | POS (48) | 2E-06 | – |
| INF344 | 8 | WT | 21 | WT | 256 | 17 | POS (48) | 4E-06 | 9E-07 |
| INF079 | 8 | WT | 20 | WT | 256 | 18 | POS (48) | 8E-07 | – |
| INF018 | 8 | WT | 21 | WT | 256 | 15 | POS (48) | 1E-06 | 4E-06 |
| INF171 | 16 | WT | 24 | WT | 256 | 13 | POS (48) | 3E-06 | 1E-06 |
| INF142 | 32 | WT | 22 | WT | 256 | 16 | POS (72) | 3E-06 | 1E-05 |
| INF223 | 32 | WT | 22 | WT | 128 | 14 | POS (24) | 5E-07 | – |
| INF161 | 32 | WT | 20 | WT | 256 | 16 | POS (48) | 1E-05 | 4E-06 |
| INF348g | 128 | non-WT | 11 | non-WT | 512 | no zone | POS (24) | 3E-05 | 8E-06 |
| INF249g | >1024 | non-WT | no zone | non-WT | >1024 | no zone | POS (24) | 1E+00 | 7E-01 |
| ATCC | |||||||||
| 13883 | 64 | WT | 21 | WT | 1024 | 20 | POS (48) | 1E-05 | 1E-05 |
| Strain # . | Standard susceptibility testing . | Heteroresistance . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| AD MIC (mg/L)a . | DD (inhibition zone, mm)b . | High inoculum AD (mg/L)c . | High inoculum DD (mm)d . | Disc elution (time, h)e . | RSP proportion in CAMHBf . | RSP proportion in SHUf . | |||
| Clinical isolates | |||||||||
| INF014 | 2 | WT | 25 | WT | 64 | 22 | POS (72) | 1E-07 | – |
| INF321 | 2 | WT | 24 | WT | 64 | 22 | POS (48) | 1E-06 | – |
| INF215 | 4 | WT | 24 | WT | 64 | 17 | POS (48) | 1E-07 | – |
| INF174 | 4 | WT | 23 | WT | 128 | 19 | POS (48) | 4E-07 | – |
| INF044 | 8 | WT | 23 | WT | 256 | 19 | POS (48) | 2E-06 | – |
| INF344 | 8 | WT | 21 | WT | 256 | 17 | POS (48) | 4E-06 | 9E-07 |
| INF079 | 8 | WT | 20 | WT | 256 | 18 | POS (48) | 8E-07 | – |
| INF018 | 8 | WT | 21 | WT | 256 | 15 | POS (48) | 1E-06 | 4E-06 |
| INF171 | 16 | WT | 24 | WT | 256 | 13 | POS (48) | 3E-06 | 1E-06 |
| INF142 | 32 | WT | 22 | WT | 256 | 16 | POS (72) | 3E-06 | 1E-05 |
| INF223 | 32 | WT | 22 | WT | 128 | 14 | POS (24) | 5E-07 | – |
| INF161 | 32 | WT | 20 | WT | 256 | 16 | POS (48) | 1E-05 | 4E-06 |
| INF348g | 128 | non-WT | 11 | non-WT | 512 | no zone | POS (24) | 3E-05 | 8E-06 |
| INF249g | >1024 | non-WT | no zone | non-WT | >1024 | no zone | POS (24) | 1E+00 | 7E-01 |
| ATCC | |||||||||
| 13883 | 64 | WT | 21 | WT | 1024 | 20 | POS (48) | 1E-05 | 1E-05 |
AD, agar dilution; DD, disc diffusion; RSP, resistant subpopulation; –, indicates resistant growth not detected after 24 h incubation.
AD was performed in triplicate (median value reported). An MIC ≤64 mg/L was used to distinguish WT from the non-WT strains.
DD was performed in triplicate (mean inhibition diameter reported). An inhibition diameter of >15 mm was used to distinguish WT from the non-WT strains. DD results applied the EUCAST reading guide that ignores any inner colonies within the inhibition zone.
AD performed with 1 × 106 cfu/spot.
DD performed with a lawn culture prepared directly using turbid overnight growth in CAMHB. Inhibition diameter ignores inner colonies within the inhibition zone.
Disc elution screening test with six FOT200 discs (Oxoid Ltd/Thermo Fisher Scientific, UK) added to 2 mL CAMHB, inoculated with 100 μL of a 109 cfu/mL bacterial suspension from an overnight culture in CAMHB, and assessed for turbidity (POS, positive) over 72 h incubation.
RSP was determined after 24 h incubation in the bladder infection model in CAMHB with G6P, or SHU, with an inflow rate of 20 mL/h and 4-hourly voiding. The proportion was calculated from growth quantified on MHA with 512 mg/L fosfomycin (and 25 mg/L G6P) compared with total growth on drug-free MHA. All RSPs were confirmed to have a fosfomycin MIC of ≥1024 mg/L by standard AD methodology, except for INF174 that measured MIC 512 mg/L.
Non-WT isolates (INF249 and INF348) both had a premature STOP codon in uhpB.
Baseline fosfomycin susceptibility and heteroresistance of the representative K. pneumoniae isolates
| Strain # . | Standard susceptibility testing . | Heteroresistance . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| AD MIC (mg/L)a . | DD (inhibition zone, mm)b . | High inoculum AD (mg/L)c . | High inoculum DD (mm)d . | Disc elution (time, h)e . | RSP proportion in CAMHBf . | RSP proportion in SHUf . | |||
| Clinical isolates | |||||||||
| INF014 | 2 | WT | 25 | WT | 64 | 22 | POS (72) | 1E-07 | – |
| INF321 | 2 | WT | 24 | WT | 64 | 22 | POS (48) | 1E-06 | – |
| INF215 | 4 | WT | 24 | WT | 64 | 17 | POS (48) | 1E-07 | – |
| INF174 | 4 | WT | 23 | WT | 128 | 19 | POS (48) | 4E-07 | – |
| INF044 | 8 | WT | 23 | WT | 256 | 19 | POS (48) | 2E-06 | – |
| INF344 | 8 | WT | 21 | WT | 256 | 17 | POS (48) | 4E-06 | 9E-07 |
| INF079 | 8 | WT | 20 | WT | 256 | 18 | POS (48) | 8E-07 | – |
| INF018 | 8 | WT | 21 | WT | 256 | 15 | POS (48) | 1E-06 | 4E-06 |
| INF171 | 16 | WT | 24 | WT | 256 | 13 | POS (48) | 3E-06 | 1E-06 |
| INF142 | 32 | WT | 22 | WT | 256 | 16 | POS (72) | 3E-06 | 1E-05 |
| INF223 | 32 | WT | 22 | WT | 128 | 14 | POS (24) | 5E-07 | – |
| INF161 | 32 | WT | 20 | WT | 256 | 16 | POS (48) | 1E-05 | 4E-06 |
| INF348g | 128 | non-WT | 11 | non-WT | 512 | no zone | POS (24) | 3E-05 | 8E-06 |
| INF249g | >1024 | non-WT | no zone | non-WT | >1024 | no zone | POS (24) | 1E+00 | 7E-01 |
| ATCC | |||||||||
| 13883 | 64 | WT | 21 | WT | 1024 | 20 | POS (48) | 1E-05 | 1E-05 |
| Strain # . | Standard susceptibility testing . | Heteroresistance . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| AD MIC (mg/L)a . | DD (inhibition zone, mm)b . | High inoculum AD (mg/L)c . | High inoculum DD (mm)d . | Disc elution (time, h)e . | RSP proportion in CAMHBf . | RSP proportion in SHUf . | |||
| Clinical isolates | |||||||||
| INF014 | 2 | WT | 25 | WT | 64 | 22 | POS (72) | 1E-07 | – |
| INF321 | 2 | WT | 24 | WT | 64 | 22 | POS (48) | 1E-06 | – |
| INF215 | 4 | WT | 24 | WT | 64 | 17 | POS (48) | 1E-07 | – |
| INF174 | 4 | WT | 23 | WT | 128 | 19 | POS (48) | 4E-07 | – |
| INF044 | 8 | WT | 23 | WT | 256 | 19 | POS (48) | 2E-06 | – |
| INF344 | 8 | WT | 21 | WT | 256 | 17 | POS (48) | 4E-06 | 9E-07 |
| INF079 | 8 | WT | 20 | WT | 256 | 18 | POS (48) | 8E-07 | – |
| INF018 | 8 | WT | 21 | WT | 256 | 15 | POS (48) | 1E-06 | 4E-06 |
| INF171 | 16 | WT | 24 | WT | 256 | 13 | POS (48) | 3E-06 | 1E-06 |
| INF142 | 32 | WT | 22 | WT | 256 | 16 | POS (72) | 3E-06 | 1E-05 |
| INF223 | 32 | WT | 22 | WT | 128 | 14 | POS (24) | 5E-07 | – |
| INF161 | 32 | WT | 20 | WT | 256 | 16 | POS (48) | 1E-05 | 4E-06 |
| INF348g | 128 | non-WT | 11 | non-WT | 512 | no zone | POS (24) | 3E-05 | 8E-06 |
| INF249g | >1024 | non-WT | no zone | non-WT | >1024 | no zone | POS (24) | 1E+00 | 7E-01 |
| ATCC | |||||||||
| 13883 | 64 | WT | 21 | WT | 1024 | 20 | POS (48) | 1E-05 | 1E-05 |
AD, agar dilution; DD, disc diffusion; RSP, resistant subpopulation; –, indicates resistant growth not detected after 24 h incubation.
AD was performed in triplicate (median value reported). An MIC ≤64 mg/L was used to distinguish WT from the non-WT strains.
DD was performed in triplicate (mean inhibition diameter reported). An inhibition diameter of >15 mm was used to distinguish WT from the non-WT strains. DD results applied the EUCAST reading guide that ignores any inner colonies within the inhibition zone.
AD performed with 1 × 106 cfu/spot.
DD performed with a lawn culture prepared directly using turbid overnight growth in CAMHB. Inhibition diameter ignores inner colonies within the inhibition zone.
Disc elution screening test with six FOT200 discs (Oxoid Ltd/Thermo Fisher Scientific, UK) added to 2 mL CAMHB, inoculated with 100 μL of a 109 cfu/mL bacterial suspension from an overnight culture in CAMHB, and assessed for turbidity (POS, positive) over 72 h incubation.
RSP was determined after 24 h incubation in the bladder infection model in CAMHB with G6P, or SHU, with an inflow rate of 20 mL/h and 4-hourly voiding. The proportion was calculated from growth quantified on MHA with 512 mg/L fosfomycin (and 25 mg/L G6P) compared with total growth on drug-free MHA. All RSPs were confirmed to have a fosfomycin MIC of ≥1024 mg/L by standard AD methodology, except for INF174 that measured MIC 512 mg/L.
Non-WT isolates (INF249 and INF348) both had a premature STOP codon in uhpB.
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