Her brother, 4 years and 7 months older, was diagnosed with CF after her diagnosis. He was born at term without complications and had a past history of frequent vomiting and poor weight gain since his first months of life. He was hospitalized for dehydration at 3 months of age and for enterorrhagia by Meckel's diverticulum at 6 months of age. At 2 years of age, recurrent upper respiratory tract infections started. Despite a voracious appetite, his difficulty in weight gain worsened. In the first consultation at the CF reference centre, at 5 years and 10 months of age, his parents reported that he had frequent coughing. On physical examination, he exhibited pallor, with a weight of 15.7 kg (2nd percentile for age), height of 109 cm (11th percentile for age), BMI of 13.3 kg/m2 (3rd percentile for age), and z scores of -1.22, -1.94 and -1.81, respectively. From the first consultation, P. aeruginosa and S. aureus respiratory chronic colonization were detected and, from the following month, also A. xylosoxidans. Attempts to eradicate P. aeruginosa also failed. Regarding sporadic colonization, the boy had two positive cultures for Haemophilus. Until 18 years of age, he had 3 exacerbations of CF treated with hospitalization for 14 days each during the 7th and 8th years and another hospitalization for viral encephalitis at age 11. In the last year, at 17 years and 6 months old, liver disease was detected, and at 18 years of age he was admitted twice: once for vasculitis and another for exacerbation of CF. However, their mother claimed that she could not understand the greater debilitation of her daughter's health compared with her son's, despite the daughter's better compliance with the treatment regimen.
The comparisons of lung function (forced expiratory volume in 1 s - FEV1), BMI and Bhalla CT score in two siblings were done at the same age and are presented in Fig. 1. Although both siblings exhibited some improvement in their absolute BMI during follow-up at the reference centre, the female's values were significantly lower than the male's (Mann-Whitney test, p < 0.0001; interquartile Range (IQR) - male: 2.5 kg/m2, female: 1.1 kg/m2).
To measure lung structural damage, 2 radiologists who were blinded to any other information reviewed the CT scans separately. They assigned modified Bhalla scores for each CT scan and reassigned the scores 1 month later. The median of the final Bhalla scores was obtained for each available CT scan. The female had lower Bhalla scores than her brother: 18.7 (1 year and 6 months), 9 (7 year and 2 months), 6.7 (10 years and 11 months) and 6.5 (13 years and 6 months). His scores were 16.5 (5 years and 10 months), 12.2 (9 years and 7 months) and 10.2 (14 years).
Their bacteriological backgrounds were studied between 3.3 and 6.5 years of age for the female and 7.7 and 9.9 years of age for her brother. Species identification was performed by conventional methods, and genotype analysis was performed by multi-locus sequence typing (MLST) of 7 housekeeping genes (nusA, rpoB, eno, gltB, lepA, nuoL, nrdA), as previously described. Allelic profiles and ST were analysed according to the PubMLST Website (http://pubmlst.org/achromobacter/). All isolates were A. xylosoxidans and belonged to the same sequence type (ST 201). This is a new ST previously identified in Brazilian CF patients by our group and added to the PubMLST Website.
The determination of minimal inhibitory concentrations (MIC) of antibiotics [ceftazidime (CAZ), ciprofloxacin (CIP), imipenem (IMP) and thimethoprim-sulfamethoxazole (TMP-SXT)] was performed using E-test strips (AB Biodisk, Solna, Sweden). The breakpoints used were those recommended by CLSI for non-Enterobacteriaceae. The isolates were considered resistant when MIC >=32 mug/mL for CAZ; MIC >=4 mug/mL for CIP; MIC >=16 mug/mL for IMP and MIC >=4/76 mug/mL for TMP-SXT. The majority of isolates (58.3%) were susceptible to all antibiotics tested (95%CI: 36.5-80.1%). All isolates were susceptible to IMP and CAZ (Fig. 2).
The swimming and swarming motility abilities were determined as described by Rashid et al.. The results are presented as the median diameters obtained in 3 independent assays performed in triplicate (Fig. 2). There were no differences in swimming phenotype between the isolates (unpaired t-test, p = 0.07; 95%CI - male: 38.1-58.2 mm, female: 27.2-45.4 mm), but the swarming phenotype was significantly higher for the A. xylosoxidans isolates recovered from the male (Mann-Whitney test, p = 0.004; IQR - male: 23 mm, female: 20 mm).
Biofilm formation on abiotic plastic surfaces was determined as described by Tendolkar et al.. Then, the A. xylosoxidans isolates were divided into different biofilm-producer classes: N, no biofilm producer; W, weak biofilm producer; M, moderate biofilm producer; and S, strong biofilm producer, as described by Stepanovic et al.. All isolates produced biofilms, and the majority were strong producers, but no significant difference in the amount of biofilm formation was detected when isolates from both patients were compared.
Data were analysed with Graph Pad Prism software, version 5.0. The specific tests used are described in the text. Significance was accepted at the p < 0.05 level.