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Cystic fibrosis (CF) is characterized by early structural lung disease caused by pulmonary infections. The nasopharynx of infants is a major ecological reservoir of potential respiratory pathogens. To investigate the development of nasopharyngeal microbiota profiles in infants with CF compared with those of healthy control subjects during the first 6 months of life. We conducted a prospective cohort study, from the time of diagnosis onward, in which we collected questionnaires and 324 nasopharynx samples from 20 infants with CF and 45 age-matched healthy control subjects. Microbiota profiles were characterized by 16S ribosomal RNA-based sequencing. We observed significant differences in microbial community composition (P < 0.0002 by permutational multivariate analysis of variance) and development between groups. In infants with CF, early Staphylococcus aureus and, to a lesser extent, Corynebacterium spp. and Moraxella spp. dominance were followed by a switch to Streptococcus mitis predominance after 3 months of age. In control subjects, Moraxella spp. enrichment occurred throughout the first 6 months of life. In a multivariate analysis, S. aureus, S. mitis, Corynebacterium accolens, and bacilli were significantly more abundant in infants with CF, whereas Moraxella spp., Corynebacterium pseudodiphtericum and Corynebacterium propinquum and Haemophilus influenzae were significantly more abundant in control subjects, after correction for age, antibiotic use, and respiratory symptoms. Antibiotic use was independently associated with increased colonization of gram-negative bacteria such as Burkholderia spp. and members of the Enterobacteriaceae bacteria family and reduced colonization of potential beneficial commensals. From diagnosis onward, we observed distinct patterns of nasopharyngeal microbiota development in infants with CF under 6 months of age compared with control subjects and a marked effect of antibiotic therapy leading toward a gram-negative microbial composition.

We report on a patient in China with no underlying illnesses who died of Burkholderia thailandensis infection despite timely treatment. This case challenges the perception that B. thailandensis is nonlethal or has low virulence. Increased clinical awareness and prompt diagnosis are essential for managing B. thailandensis infections and preventing fatal outcomes.

It was recently proposed that bactericidal antibiotics, besides through specific drug-target interactions, kill bacteria by a common mechanism involving the production of reactive oxygen species (ROS). However, this mechanism involving the production of hydroxyl radicals has become the subject of a lot of debate. Since the contribution of ROS to antibiotic mediated killing most likely depends on the conditions, differences in experimental procedures are expected to be at the basis of the conflicting results. In the present study different methods (ROS specific stainings, gene-expression analyses, electron paramagnetic resonance, genetic and phenotypic experiments, detection of protein carbonylation and DNA oxidation) to measure the production of ROS upon antibiotic treatment in Burkholderia cepacia complex (Bcc) bacteria were compared. Different classes of antibiotics (tobramycin, ciprofloxacin, meropenem) were included, and both planktonic and biofilm cultures were studied. Our results indicate that some of the methods investigated were not sensitive enough to measure antibiotic induced production of ROS, including the spectrophotometric detection of protein carbonylation. Secondly, other methods were found to be useful only in specific conditions. For example, an increase in the expression of OxyR was measured in Burkholderia cenocepacia K56-2 after treatment with ciprofloxacin or meropenem (both in biofilms and planktonic cultures) but not after treatment with tobramycin. In addition results vary with the experimental conditions and the species tested. Nevertheless our data strongly suggest that ROS contribute to antibiotic mediated killing in Bcc species and that enhancing ROS production or interfering with the protection against ROS may form a novel strategy to improve antibiotic treatment.

Burkholderia semiarida was previously identified solely as a plant pathogen within the Burkholderia cepacia complex. We present a case in China involving recurrent pneumonia attributed to B. semiarida infection. Of note, the infection manifested in an immunocompetent patient with no associated primary diseases and endured for >3 years.

Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) can cause serious nosocomial infections. The reported misidentification of Bcc as P. aeruginosa , besides the contrasting susceptibility patterns between these two pathogens, underscores the importance of accurate identification and the need to explore therapeutic approaches that could eradicate them both. Our study aimed to evaluate the susceptibilities of both pathogens to different antibiotics and test the effectiveness of different antimicrobial combinations. In addition, we compared conventional phenotypic and automated identification methods using the VITEK 2 system with PCR amplification and sequencing of recA gene to differentiate between P. aeruginosa and Bcc. Amplification by PCR of the recA gene showed high specificity in the identification of Bcc in comparison with conventional and automated phenotypic methods. Significant differences in susceptibility were observed with P. aeruginosa isolates having more susceptibility to colistin and ceftazidime, while Burkholderia isolates were more susceptible to cotrimoxazole and minocycline. Both P. aeruginosa and Burkholderia isolates tested were more susceptible to N-Acetyl-L-Cysteine (NALC) plus meropenem or levofloxacin, followed by gentamicin plus piperacillin/tazobactam. Synergistic effect of NALC plus meropenem or levofloxacin may help in the eradication of both pathogens to overcome the diagnostic challenges posed by the possible misidentification using conventional identification methods.

Background. Burkholderia cepacia complex (Bcc) are a group of multidrug-resistant gram-negative bacteria rarely reported in patients without cystic fibrosis (CF) or immunocompromising conditions. We investigated Bcc bloodstream infections (BSIs) in a cohort of non-CF patients from the US Veterans Health Administration (VHA). Methods. Using VHA databases, we identified patients with Bcc BSI at facilities nationwide from 1999 through 2015. We ascertained clinical characteristics, treatments, and outcomes and identified factors associated with 30-day mortality in logistic regression analysis. Results. We identified 248 patients with Bcc BSI, who were of advanced age (mean, 68 years), chronically ill, and had severe disease. The most common sources were central venous catheters (41%) and pneumonia (20%). Most cases were hospital-acquired (155 [62%]) or healthcare-associated (70 [28%]). Mortality at 14, 30, and 90 days was 16%, 25%, and 36%, respectively. Trimethoprimsulfamethoxazole (TMP-SMX) and fluoroquinolones were active against 94% and 88% of isolates, respectively. Susceptibility to ceftazidime and meropenem occurred in approximately 70% of the isolates. The most prescribed antibiotics were fluoroquinolones (35%), followed by carbapenems (20%), TMP-SMX (18.5%), and ceftazidime (11%). In regression analysis, age (OR, 1.06 [95% confidence interval {CI}, 1.02–1.10], per added year) and the Pitt bacteremia score (OR, 1.65 [95% CI, 1.44–1.94], per unit increase) were associated with higher 30-day mortality. Conclusions. In this large cohort of BSIs caused by Bcc, cases were mostly hospital-acquired and we observed high mortality, significant resistance to ceftazidime, and limited use of TMP-SMX. These observations add to our understanding of Bcc infection in non-CF patients and highlight the need for interventions to improve their outcome.

The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite. We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an \"ortholog rescue\" strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail. This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.

Background Cervical suppurative lymphadenitis in children is commonly caused by Staphylococcus aureus or Streptococcus pyogenes. However, cases caused by Burkholderia multivorans (BM) are rare. The clinical presentation lacks specificity, making it difficult for clinicians to recognize, which may delay diagnosis and treatment. Case presentation We report a case of a 5-year-old boy admitted with recurrent fever and neck swelling. Initial treatment with meropenem and linezolid was ineffective, and symptoms persisted after 24 days of conservative therapy. Aspiration of pus yielded negative culture results. Definitive diagnosis was achieved through surgical biopsy of cervical lymph nodes, pathological examination, and metagenomic next-generation sequencing (mNGS), which identified BM as the causative pathogen. The patient was successfully treated with a combination of trimethoprim-sulfamethoxazole and meropenem. The cervical lesion exhibited granulomatous inflammation and was managed with adjunctive vacuum-assisted closure (VAC) therapy, resulting in complete wound healing without recurrence. Conclusions This study aims to raise awareness among all specialists about BM as a potential causative agent in cervical suppurative lymphadenitis. Early recognition and timely intervention can reduce misdiagnosis and missed diagnoses, improving patient outcomes.

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.

Objective To report the clinical presentation and outcomes of cluster postcataract Burkholderia cepacia complex endophthalmitis, the source of infection and clonal relatedness of the isolates. Methods This was a retrospective study on 13 patients who developed acute postoperative endophthalmitis, along with an infiltrate at the corneal section, after an uneventful cataract surgery with intraocular lens implantation. Aqueous aspirates, vitreous aspirates and environmental surveillance specimens were sampled. Genotypic diversity was determined by PCR using BOX-PCR for each strain, and the clonal relationship was established between clinical and eye drops isolates. Results Vitreous samples showed B. cepacia in cultures in all 13 eyes. Among the samples from various surveillance specimens cultured, topical anaesthetic eye drops grew B. cepacia. The isolates from the patients and the eye drops solution revealed matching banding patterns in BOX-PCR. Isolates from the patients and eye drops were susceptible to cefotaxime and piperacillin/tazobactam only. 9 (69%) patients out of 13 had a final visual acuity of 6/60 or better. Among the remaining four patients, three had a vision of perception of light and one had final vision of 1/60. Conclusions Microbiology culture and BOX-PCR results revealed contamination of local anaesthetic eye drops and the same organism was cultured from a group of patients with acute-onset postoperative endophthalmitis after an uneventful cataract surgery. Outbreaks may occur in the most vigilant settings, and any sterile consumable may be a common link.