Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient.

BACKGROUND: Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. OBJECTIVES: We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. METHODS: Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. RESULTS: P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. CONCLUSIONS: A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

Enterovirus D-68 in children presenting for acute care in the hospital setting.

BACKGROUND: Severe respiratory disease associated with enterovirus D68 (EV-D68) has been reported in hospitalized pediatric patients. Virologic and clinical characteristics of EV-D68 infections exclusively in patients presenting to a hospital Emergency Department (ED) or urgent care have not been well defined. METHODS: Mid-nasal swabs from pediatric patients with respiratory symptoms presenting to the ED or urgent care were evaluated using a commercial multiplex PCR platform. Specimens positive for rhinovirus/enterovirus (HRV/EV) were subsequently tested using real-time reverse-transcriptase PCR for EV-D68. The PCR cycle threshold (CT) was used as a viral load proxy. Clinical outcomes were compared between patients with EV-D68 and patients without EV-D68 who tested positive for HRV/EV. RESULTS: From August to December 2014, 511 swabs from patients with HRV/EV were available. EV-D68 was detected in 170 (33%) HRV/EV-positive samples. In multivariable models adjusted for age and underlying asthma, patients with EV-D68 were more likely to require hospitalization for respiratory reasons (odds ratio (OR): 3.11, CI: 1.85-5.25), require respiratory support (OR: 1.69, CI: 1.09-2.62), have confirmed/probable lower respiratory tract infection (LRTI; OR: 3.78, CI: 2.03-7.04), and require continuous albuterol or steroids (OR: 3.91, CI: 2.22-6.88 and OR: 4.73, CI: 2.65-8.46, respectively). Higher EV-D68 viral load was associated with need for respiratory support and LRTI in multivariate models. CONCLUSIONS: Among pediatric patients presenting to the ED or urgent care, EV-D68 causes more severe disease than non-EV-D68 HRV/EV independent of underlying asthma. High viral load was associated with worse clinical outcomes. Rapid and quantitative viral testing may help identify and risk stratify patients.

Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples.

Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx1, stx2, and rfbEO157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbEO157, and LD-PCR results prompted successful recovery of E. coli O157 (n = 25) and non-O157 STEC (n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid "mega"-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

Changes in cystic fibrosis sputum microbiology in the United States between 1995 and 2008.

STUDY OBJECTIVES: The study objective was to identify changes in cystic fibrosis (CF) sputum microbiology over 13 years. METHODS: This study recruited a contemporary cohort of CF patients meeting similar eligibility criteria as the 520 subjects in the Phase 3 trials of inhaled tobramycin (historical cohort). Subjects submitted a single sputum specimen to a centralized laboratory for culture and susceptibility testing. Data were summarized and cross-sectional prevalence estimates were compared between cohorts. Exploratory analyses examined associations between recent antibiotic exposures and resistance prevalence. RESULTS: Sputum samples from 267 subjects from 33 US CF centers were submitted for testing. A total of 656 Pseudomonas aeruginosa isolates were identified from 253 culture-positive subjects. Comparison between cohorts revealed an increase in the prevalence of subjects with tobramycin resistant (11.8% vs. 30.4%, P < 0.001) and amikacin resistant (24.2% vs. 42.7%, P < 0.001) P. aeruginosa. Prevalence of ciprofloxacin resistance was similar (34.4% vs. 33.6%, P = 0.81). Within the contemporary cohort, potential associations between recent antibiotic exposures and prevalence of P. aeruginosa resistance were examined; findings included that exposure to intravenous carbapenems was significantly associated with aztreonam resistance, meropenem resistance, and multidrug resistance (P = 0.0002, P = 0.0003, and P = 0.0002, respectively). Prevalences of Staphylococcus aureus, methicillin-resistant S. aureus, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans were also increased in the contemporary cohort. CONCLUSIONS: We identified important changes in the patterns of CF airway microbiology, including increased aminoglycoside resistance and prevalence of other antibiotic resistant organisms. These changes are concerning to clinicians caring for individuals with CF because they impact treatment options. These data point to a critical need to develop new antimicrobials for CF.