Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.
Methicillin-resistant Staphylococcus aureus (MRSA) colonization in cystic fibrosis (CF) patients is an increasing problem in many countries. In our Respiratory Center at the Hospital de Niños "Dr. Ricardo Gutiérrez", Buenos Aires, Argentina, the prevalence has climbed from 23% in 1995 up to 32% in 2011. Our objective was to analyze the diversity of MRSA isolates recovered from respiratory samples of CF patients attending our center, characterizing their phenotypes and clonal distribution. Therefore, a prospective study was conducted on all CF patients attending the pediatric Respiratory Center between June 2012 and May 2013 to collect MRSA isolates. Antibiotic susceptibility testing, multilocus sequence typing, pulsed-field gel electrophoresis, spa typing, and agr genotyping were performed on collected isolates. The prevalence of MRSA during this period was 34.2%, and 71.9% of the patients were infected with isolates that carried SCCmec IV. High resistance rates were detected for gentamicin, erythromycin, clindamycin, ciprofloxacin, and rifampicin. Strains related to the community-associated MRSA clones, ST5-IV and ST30-IV, were the most frequently recovered. Remarkably, even though most of the isolates were related to these clones, the rate of multi-resistance shown in CF patients was higher than that reported for the same lineages recovered from other infections in our country.
Species of the genus Scedosporium (family Microascaceae, phylum Ascomycota) are responsible for a wide range of opportunistic human infections, and have a low susceptibility to most antifungal drugs. It is well known that the pattern of Scedosporium species distribution varies according to geographic region. To assess the diversity of Scedosporium species in Argentina involved in human infections, we carried out a retrospective study reviewing 49 strains from clinical samples sent for diagnosis to the National Clinical Mycology Reference Laboratory between 1985 and 2019. Then, a phenotypic characterization, a phylogenetic study and and in vitro susceptibility test to antifungals were carried out. An analysis of combined nucleotide sequences dataset of the internal transcribed spacer of the ribosomal DNA (ITS) and of a fragment of the β-tubulin gene (BT2) demonstrated that 92 % of the strains belonged to the species S. boydii, S. apiospermum and S. angustum, all them pertaining to S. apiospermum species complex. However, two strains (4%) were identified as S. aurantiacum, a species never reported in clinical settings in the Americas’. Surprisingly, one of them displayed a polycytella-like conidiogenesis, up to date only reported for S. apiospermum. In addition, the strain DMic 165285 was phylogenetically located far away from the rest of the species, so is proposed as the novel species Scedosporium americanum. On the other hand, from all seven antifungals tested, voriconazole and posaconazole were the most active drugs against Scedosporium spp.
Staphylococcus aureus chronic airway infection in patients with cystic fibrosis (CF) allows this pathogen to adapt over time in response to different selection pressures. We have previously shown that the main sequence types related to community-acquired methicillin-resistant S. aureus (MRSA) infections in Argentina – ST5 and ST30 – are also frequently isolated from the sputum of patients with CF, but in these patients they usually display multi-drug antimicrobial resistance. In this study, we sequenced the genomes of MRSA from four paediatric CF patients with the goal of identifying mutations among sequential isolates, especially those possibly related to antimicrobial resistance and virulence, which might contribute to the adaptation of the pathogen in the airways of patients with CF. Our results revealed genetic differences in sequential MRSA strains isolated from patients with CF in both their core and accessory genomes. Although the genetic adaptation of S. aureus was distinct in different hosts, we detected independent mutations in thyA, htrA, rpsJ and gyrA – which are known to have crucial roles in S. aureus virulence and antimicrobial resistance – in isolates recovered from multiple patients. Moreover, we identified allelic variants that were detected in all of the isolates recovered after a certain time point; these non-synonymous mutations were in genes associated with antimicrobial resistance, virulence, iron scavenging and oxidative stress resistance. In conclusion, our results provide evidence of genetic variability among sequential MRSA isolates that could be implicated in the adaptation of these strains during chronic CF airway infection.
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