Burkholderia cenocepacia is a Gram-negative aerobic bacterium that belongs to a group of opportunistic pathogens displaying diverse environmental and pathogenic lifestyles. B. cenocepacia is known for its ability to cause lung infections in people with cystic fibrosis and it possesses a large 8 Mb multireplicon genome encoding a wide array of pathogenicity and fitness genes. Transcriptomic profiling across nine growth conditions was performed to identify the global gene expression changes made when B. cenocepacia changes niches from an environmental lifestyle to infection. In comparison to exponential growth, the results demonstrated that B. cenocepacia changes expression of over one-quarter of its genome during conditions of growth arrest, stationary phase and surprisingly, under reduced oxygen concentrations (6% instead of 20.9% normal atmospheric conditions). Multiple virulence factors are upregulated during these growth arrest conditions. A unique discovery from the comparative expression analysis was the identification of a distinct, co-regulated 50-gene cluster that was significantly upregulated during growth under low oxygen conditions. This gene cluster was designated the low-oxygen-activated (lxa) locus and encodes six universal stress proteins and proteins predicted to be involved in metabolism, transport, electron transfer and regulation. Deletion of the lxa locus resulted in B. cenocepacia mutants with aerobic growth deficiencies in minimal medium and compromised viability after prolonged incubation in the absence of oxygen. In summary, transcriptomic profiling of B. cenocepacia revealed an unexpected ability of aerobic Burkholderia to persist in the absence of oxygen and identified the novel lxa locus as key determinant of this important ecophysiological trait.

f Respiratory infection in cystic fibrosis (CF) is polymicrobial, but standard sputum microbiology does not account for the lung microbiome or detect changes in microbial diversity associated with disease. As a clinically applicable CF microbiome surveillance scheme, total sputum nucleic acids isolated by a standard high-throughput robotic method for accredited viral diagnosis were profiled for bacterial diversity using ribosomal intergenic spacer analysis (RISA) PCR. Conventional culture and RISA were performed on 200 paired sputum samples from 93 CF adults; pyrosequencing of the 16S rRNA gene was applied to 59 patients to systematically determine bacterial diversity. Compared to the microbiology data, RISA profiles clustered into two groups: the emerging nonfermenting Gram-negative organisms (eNFGN) and Pseudomonas groups. Patients who were culture positive for Burkholderia, Achromobacter, Stenotrophomonas, and Ralstonia clustered within the eNFGN group. Pseudomonas group RISA profiles were associated with Pseudomonas aeruginosa culture-positive patients. Sequence analysis confirmed the abundance of eNFGN genera and Pseudomonas within these respective groups. Low bacterial diversity was associated with severe lung disease (P < 0.001) and the presence of Burkholderia (P < 0.001). An absence of Streptococcus (P < 0.05) occurred in individuals with lung function in the lowest quartile. In summary, nucleic acids isolated from CF sputum can serve as a single template for both molecular virology and bacteriology, with a RISA PCR rapidly detecting the presence of dominant eNFGN pathogens or P. aeruginosa missed by culture (11% of cases). We provide guidance for how this straightforward CF microbiota profiling scheme may be adopted by clinical laboratories. C ystic fibrosis (CF) is an inherited condition characterized by chronic endobronchial infection leading eventually to respiratory failure (1). Traditional culture-based microbiological techniques readily identify pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, which are common and typical of CF infection (2). Other CF pathogens, such as Burkholderia cepacia complex and emerging pathogens such as Achromobacter, Stenotrophomonas, Ralstonia, and Pandoraea species, are a challenge for conventional microbiology due to their taxonomic complexity (2). These emerging non-Pseudomonas, nonfermenting Gram-negative (eNFGN) species present multiple problems for people with CF, including innate antibiotic resistance, uncertain pathogenic outcome, and potential for transmission, and are possible contraindicators for subsequent lung transplantation (3).CF therapy has evolved to optimize the prevention and suppression of bacterial infections known to be associated with clinical deterioration, such as P. aeruginosa (4, 5) or B. cepacia complex (6). Recent culture-independent analysis of CF infection revealed the presence of considerable bacterial diversity (collectively known as the CF microbiota) that is not captured by standard culture (7). Facultative and obl...

Significance and Impact of the Study: The widespread presence of biofilms on dry surfaces in healthcare settings has been recently documented. These dry surface biofilms (DSB) present an unprecedented challenge to cleaning and disinfection processes. Here, we describe a practical efficacy protocol based on an in vitro Staphylococcus aureus DSB model. The protocol measures reduction in viability, transferability and biofilm regrowth post-treatment to provide altogether a practical assessment of product efficacy against dry surface biofilms. AbstractDry surface biofilms (DSB) harbouring pathogens are widespread in healthcare settings, are difficult to detect and are resistant to cleaning and disinfection interventions. Here, we describe a practical test protocol to palliate the lack of standard efficacy test methods for DSB. Staphylococcus aureus DSB were produced over a 12-day period, grown with or without the presence of organic matter, and their composition and viability were evaluated. Disinfectant treatment was conducted with a modified ASTM2967-15 test and reduction in viability, transferability and biofilm regrowth post-treatment were measured. Dry surface biofilms produced over a 12-day period had a similar carbohydrates, proteins and DNA content, regardless of the presence or absence of organic matter. The combination of sodium hypochlorite (1000 ppm) and a microfiber cloth was only effective against DSB in the absence of organic load. With the increasing concerns of the uncontrolled presence of DSB in healthcare settings, the development of effective intervention model in the presence of organic load is appropriate for the testing of biocidal products, while the use of three parameters, log 10 reduction, transferability and regrowth, provides an accurate and practical measurement of product efficacy.Staphylococcus aureus dry surface biofilm model K. Ledwoch et al.

Background: Pathogens in drain biofilms pose a significant risk for hospital-acquired infection. However, the evidence of product effectiveness in controlling drain biofilm and pathogen dissemination are scarce. A novel in-vitro biofilm model was developed to address the need for a robust, reproduceable and simple testing methodology for disinfection efficacy against a complex drain biofilm. Methods: Identical complex drain biofilms were established simultaneously over 8 days, mimicking a sink trap. Reproducibility of their composition was confirmed by nextgeneration sequencing. The efficacy of sodium hypochlorite 1000 ppm (NaOCl), sodium dichloroisocyanurate 1000 ppm (NaDCC), non-ionic surfactant (NIS) and peracetic acid 4000 ppm (PAA) was explored, simulating normal sink usage conditions. Bacterial viability and recovery following a series of 15-min treatments were measured in three distinct parts of the drain. Results: The drain biofilm consisted of 119 mixed species of Gram-positive and -negative bacteria. NaOCl produced a >4 log 10 reduction in viability in the drain front section alone, while PAA achieved a >4 log 10 reduction in viability in all of the drain sections following three 15-min doses and prevented biofilm regrowth for >4 days. NIS and NaDCC failed to control the biofilm in any drain sections. Conclusions: Drains are one source of microbial pathogens in healthcare settings. Microbial biofilms are notoriously difficult to eradicate with conventional chemical biocidal products. The development of this reproducible in-vitro drain biofilm model enabled understanding of the impact of biocidal products on biofilm spatial composition and viability in different parts of the drain.

Aims: Dry surface biofilms (DSB) survive on environmental surfaces throughout hospitals, able to resist cleaning and disinfection interventions. This study aimed to produce a dual species DSB and explore the ability of commercially available wipe products to eliminate pathogens within a dual species DSB and prevent their transfer. Methods and Results:Staphylococcus aureus was grown with two different species of Bacillus on stainless steel discs, over 12 days using sequential hydration and dehydration phases. A modified version of ASTM 2967-15 was used to test six wipe products including one water control with the Fitaflex Wiperator. Staphylococcus aureus growth was inhibited when combined with Bacillus subtilis. Recovery of S. aureus on agar from a dual DSB was not always consistent. Our results did not provide evidence that Bacillus licheniformis protected S. aureus from wipe action. There was no significant difference of S. aureus elimination by antimicrobial wipes between single and dual species DSB. B. licheniformis was easily transferred by the wipe itself and to new surfaces both in a single and dual species DSB, whilst several wipe products inhibited the transfer of S. aureus from wipe. However, S. aureus direct transfer to new surfaces was not inhibited post-wiping. Conclusions:Although we observed that the dual DSB did not confer protection of S. aureus, we demonstrated that environmental species can persist on surfaces after disinfection treatment. Industries should test DSB against future products and hospitals should consider carefully the products they choose. Significance and Impact of the Study:To our knowledge, this is the first study reporting on the production of a dual species DSB. Multispecies DSB have been identified throughout the world on hospital surfaces, but many studies focus on single species biofilms. This study has shown that DSB behave differently to hydrated biofilms.