Nitrofurantoin

The plant genotype Eyach 15-2 (CS76399), collected from Eyach, Germany, was previously determined to represent a plant genetic background common to the geographical region (Bomblies et al., 2010 (link)). Seeds were sterilized by overnight incubation at −80°C, followed by 4 hours of bleach treatment at room temperature (seeds in open 2 mL tube in a desiccator containing a beaker with 40 mL Chlorox and 1 mL HCl (32%)). The seeds were stratified for three days at 4°C in the dark on ½ MS media. Plants were grown in 3-4 mL ½ MS medium in six-well plates in long-day (16 hours) at 16°C. 12-14 days after stratification, plants were infected with single bacterial strains.
Bacteria were grown overnight in Luria broth and the relevant antibiotic (either 10 μg/mL of Kanamycin or Nitrofurantoin), diluted 1:10 in the morning and grown for 2 additional hours until they entered log phase. The bacteria were pelleted at 3500 g, resuspended in 10 mM MgSO₄ to a concentration of OD₆₀₀ = 0.01. 200 μl of bacteria were drip-inoculated with a pipette onto the whole rosette. Plates were sealed with Parafilm and returned to the growth chamber. Seven days after infection, whole rosettes were cut from the plant and fresh mass was assessed.
For growth assays of dead bacteria, we performed growth and dilution of bacteria as above, then boiled the final preparation at 95°C for 38 minutes. Plants were treated with the dead bacteria in the same manner as described above.

The Kirby Bauer disk diffusion method was used to determine the antimicrobial susceptibility patterns of the E. coli isolates against a panel of 14 antimicrobial agents namely ampicillin (10 μg), amoxicillin/clavulanic acid (20/10 μg), tetracycline (30 μg), gentamicin (10 μg), cefuroxime (30 μg), streptomycin (10 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), sulfamethoxazole-trimethoprim (10 μg), nitrofurantoin (300 μg), ceftriaxone (30 μg), imipenem (10 μg), ceftazidime (30 μg) and cefotaxime (30 μg) as previously described [28 ]. Furthermore, the minimum inhibitory concentrations (MIC) against a panel of 16 antimicrobial agents for all E. coli isolates were determined by broth microdilution assay methods using the Gram-negative Sensititre™ (ESBL) plate according to the manufacturer’s instructions. These antimicrobials comprised ampicillin, cefazolin, ceftriaxone, cefepime, cefoxitin, cefotaxime, cefpodoxime, ceftazidime, cephalothin, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin/tazobactam, cefotaxime/clavulanic acid and ceftazidime/clavulanic acid. The recommendations of the Clinical and Laboratory Standards Institute (CLSI) M100 31^st Edition were used to interpret the results [29 ]. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used for internal quality control. Multidrug resistance (MDR) was defined as resistance to three or more drug classes.

GPs participating in the JGPN register a diagnosis for each consultation using International Classification of Primary Care (ICPC) codes. As a first step, medical record data were collected from all consultations of adult males with ICPC codes U01 (dysuria/painful urination), U02 (urinary frequency/urgency), U70 (pyelonephritis/pyelitis), U71 (cystitis), and Y73 (prostatitis) for which a UTI-related antibiotic (nitrofurantoin, trimethoprim, ciprofloxacin, amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, fosfomycin, or norfloxacin) was prescribed within 7 days before and after the start date. A UTI episode after a UTI consultation-free interval of 30 days was considered a new UTI episode. Second, UTI episodes were excluded if:

the patient had an indwelling urinary catheter;

no information on patient-reported symptoms and GP-assessed signs was available;

one UTI episode was associated with two prescriptions for different antibiotics on the same day;

the antibiotic prescription was for prophylactic or future use, and

the patient was anatomically female.

Next, medical records of all remaining UTI episodes were manually screened and scored for patient-reported symptoms and GP-assessed signs of a complicated UTI, namely fever reported by patients and/or assessed  by  GPs,  patient-reported  malaise  and/or cold shivers, and GP-assessed ‘clinically severely ill’, costovertebral angle tenderness, perineal pain, and/or signs of delirium.² A UTI was considered uncomplicated when all of these signs and symptoms were absent. If a sign or symptom was not recorded it was considered absent. Screening and scoring of 400 variables regarding signs and symptoms of complicated UTI was done in duplicate by two independent investigators with an agreement of 99% (kappa 0.94 [almost perfect agreement]).
The following data were extracted for each uncomplicated UTI episode: age, comorbidities (diabetes, cardiovascular, pulmonary, oncological, nephrological, urological, and neurological and immunocompromising conditions; see Supplementary Appendix S1), number and type of antibiotic prescriptions using Anatomical Therapeutic Chemical codes, and hospital referrals.
Treatment failure was defined as an antibiotic prescription for a different antibiotic >1 day after the initial prescription or an acute hospital referral to urology or internal medicine >1 day after the antibiotic prescription.
The Dutch UTI guideline recommends nitrofurantoin (first choice) or trimethoprim (second choice) for treatment of uncomplicated UTI in males.² GPs were therefore considered adherent to the guideline if they prescribed one of those antibiotics. If treatment failure had occurred in the year before the current UTI episode, ciprofloxacin, amoxicillin/clavulanic acid, and trimethoprim/sulfamethoxazole were considered adherent as well.
As data on UTI episodes from 2013 were not available, treatment failure in the year before 2014 could not be determined. The year 2014 was therefore excluded from the adherence analysis.