Study population
In this retrospective study, we reviewed all episodes of PD-related peritonitis caused by NF-GNB that occurred between June 1997 and December 2015 in a single Brazilian university center. The exclusion criteria were: episodes with incomplete clinical data, relapse (episode caused by the same species or a negative culture result within 28 days of completion of antibiotic therapy), recurrence (episode caused by other species within 28 days after starting antibiotic therapy), and repeat episode (episode caused by the same species or after 28 days following completion of antibiotic therapy)
The diagnosis of peritonitis was made when at least two of the following criteria were present: presence of a cloudy peritoneal effluent; abdominal pain; dialysate containing more than 100 leukocytes per mL (at least 50% polymorphonuclear cells); and positive culture of dialysate [23]. The outcomes were defined as: resolution (disappearance of signs and symptoms within 5 days after the initiation of antibiotic therapy); relapse, refractory peritonitis (presence of turbid dialysate after 5 days of treatment with appropriate antibiotics); peritonitis-related death (death of a patient with active peritonitis or the death of a patient who had an episode within the previous 4 weeks) [23]; and nonresolution (catheter removal before the 5th day of treatment, refractory peritonitis, relapse, or peritonitis-related death).
Catheter insertion and care and dialysis procedures
The catheter placements were made under supervision of a senior nephrologist with percutaneous blind insertion of a double cuff straight Tenckhoff catheter using the Seldinger technique. Until 2003, no patients used antibiotic cream application at the catheter exit-site; from 2003 to 2006, we prescribed daily mupirocin cream, and from January 2007 daily gentamicin was prescribed to all incident patients. All patients used a semiocclusive dressing with sterile gauze and microporous adhesive tape.
Until 1999, the CAPD connection systems were the Y set type; the twin bag was introduced in 1999. APD was introduced in 1998, and its indication and prescription were based on clinical criteria or the patient’s preference. For both PD modalities, we used standard glucose solutions, with low pH and high glucose degradation product (GDP) levels.
The diagnosis of exit-site and tunnel infection followed International Society for Peritoneal Dialysis (ISPD) criteria Exit-site infection (ESI) is the presence of purulent discharge, with or without erythema of the skin at the catheter-epidermal interface. Tunnel infection is the presence of clinical inflammation or ultrasonographic evidence of collection along the catheter tunnel.
Data collection
We recorded the following information for each case: date, preexistent ESI (ESI diagnosed until four weeks before a peritonitis episode), tunnel infection, topical antibiotic use at the catheter exit-site, initial antimicrobial treatment for peritonitis and adjustments, outcome, treatment time before the peritonitis episode (dialysis vintage) , patient’s characteristics (age, sex, race [Caucasian or non-Caucasian], underlying kidney disease, previous peritonitis by other bacteria, PD modality (continuous ambulatory PD or automated PD), and characteristics of the causative germ (species, biofilm production capability, and in vitro antibiotic susceptibility).
Culture and storage
After diagnosis, each dialysate sample was processed following the recommendations of the ISPD [23]. Cultures were performed using the Bactec System (Becton Dickinson Company, Sparks, MD) and then seeded onto blood agar if there was signaling the positivity on the cultures bottles.. After isolation, identification, and susceptibility testing, strains were stored at –70°C.
For the present study, the stored samples were reisolated on MacConkey agar plates and reidentified. For this, isolates were gram-stained to confirm purity and determine each isolate’s morphology and specific color. Afterward, the isolates were identified by conventional biochemical testing [25] and by mass spectrometry using MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) technology (MALDI-ToF VITEK® MS, Brazil) [26].
Microbiological tests
In vitro susceptibility
The in vitro susceptibility to amikacin, ciprofloxacin, cefepime, imipenem, and ceftazidime was determined by the minimum inhibitory concentration (MIC)based on gradient diffusion, using the E test ((bioMérieux, Inc., Durham, NC) The proportion of strains susceptible to each drug was defined based on the 2016 Clinical Laboratory Standards Institute breakpoints [27]. When strains presented intermediate MIC values, we considered them resistant.
Biofilm production
The bacterial samples were grown in Tryptic Soy Broth (TSB) (BD™, Le Pont de Claix, France) at 37°C for 18 hours. To assess the bacterial ability to adhere to abiotic surfaces, we used 96-well polystyrene plates and added 200 µl of TSB and 10 µ of the bacterial suspension (approximately 108 CFU/mLµ) to each well, except one well that was inoculated only with culture medium to be used as a reading standard (blank). The plates were incubated at 37°C for 48 hours and then washed with phosphate-buffered saline 4 times to remove non-adherent bacteria. Bacteria that adhered to the abiotic surface were then fixed with formalin (2%), and after 20 minutes, the formalin was removed, and the preparations were washed 4 more times with water. Then, the preparations were stained with a crystal violet solution (1%) for 20 minutes, after which they were washed 3 times with water to remove excess dye. After drying, the dye was solubilized with methanol for 10 minutes, and the optical density, measured at 540 nm, was determined [28]. Then, we classified the biofilm production into one of four categories as previously published [28]: no producer, weak producer, moderate producer, and strong producer. In our study, we opted for a 48 hours method, instead of faster methods, to obtain a more reliable result, as it allows the strains to have enough time for the production of biofilm because some NF-GNB species show slow growth.
Clinical-microbiological associations
Each patient’s characteristics, pre-existent ESI, topical antibiotic use at the catheter exit-site, initial treatment for peritonitis, treatment adjustment with two antipseudomonal antibiotics, previous peritonitis by other bacteria, and microbiological characteristics were analyzed regarding their association with the outcome. For this purpose, we classified the outcomes into two mutually exclusive results: resolution or nonresolution.
Statistical analysis
For comparison between frequencies, we used the chi-squared test or Fisher's exact test. Binary logistic regression with a backward stepwise procedure was used to determine the independent predictors of outcomes. For this purpose, we first performed a univariate logistic regression analysis to select the variables that would enter the final model, with p> 0.20 as the elimination criterion. Collinearity among variables was tested, and if statistically significant interactions occurred, one of the variables was excluded. A p value < 0.05 was considered significant.