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This study was designed to investigate the individual or combined effects of sanitizers on survival of planktonic or sessile Listeria monocytogenes cells at different phase of growth. The sanitizers tested included: (i) acetic acid (pH 5.0), (ii) NaOH (pH 12.0), (iii) 10% Na2SO4, (iv) 10% Na2SO4 and acetic acid (pH 5.0), (v) 10% Na2SO4 and NaOH (pH 12.0), (vi) a quaternary ammonium (20 ppm) and (vii) glyceryl monolaurate (75 ppm). Results revealed a great efficacy of alkaline treatments on both sessile and planktonic cells with a slightly higher resistance of 6 h biofilms. Quaternary ammonium appeared very effective in killing more than 98% of cells, but a resistance of 7 days biofilm was observed. Other sanitizers did not succeed in inhibiting totally the pathogen but acted in a similar way on both sessile and planktonic cells. Renewing the medium or not do not seem to be the major cause of a resistance emergence.

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5'-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.

Acinetobacter baumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living) and sessile (biofilm) forms of A. baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures) and sessile (biofilm) cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A. baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

Due to a high unresponsiveness to chemotherapy, biofilm formation is an important medical problem that frequently occurs during infection with many bacterial pathogens. In this study, the marine sponge-derived natural compounds 4,6-dibromo-2-(2′,4′-dibromophenoxy)phenol and 3,4,6-tribromo-2-(2′,4′-dibromophenoxy)phenol were found to exhibit broad antibacterial activity against medically relevant gram-positive and gram-negative pathogens. The compounds were not only bactericidal against both replicating and stationary phase–persistent planktonic cells of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa; they also killed biofilm-incorporated cells of both species while not affecting biofilm structural integrity. Moreover, these compounds were active against carbapenemase-producing Enterobacter sp. This simultaneous activity of compounds against different growth forms of both gram-positive and gram-negative bacteria is rare. Genome sequencing of spontaneous resistant mutants and proteome analysis suggest that resistance is mediated by downregulation of the bacterial EIIBC phosphotransferase components scrA and mtlA in MRSA likely leading to a lower uptake of the molecules. Due to their only moderate cytotoxicity against human cell lines, phenoxyphenols provide an interesting new scaffold for development of antimicrobial agents with activity against planktonic cells, persisters and biofilm-incoporated cells of ESKAPE pathogens.Key points• Brominated phenoxyphenols kill actively replicating and biofilm-incorporated bacteria.• Phosphotransferase systems mediate uptake of brominated phenoxyphenols.• Downregulation of phosphotransferase systems mediate resistance.

Bacterial growth and behavior have been studied in microgravity in the past, but little focus has been directed to cell size despite its impact on a myriad of processes, including biofilm formation, which is impactful regarding crew health. To interrogate this characteristic, supernatant aliquots of P. aeruginosa cultured on different materials and media on board the International Space Station (ISS) as part of the Space Biofilms Project were analyzed. For that experiment, P. aeruginosa was grown in microgravity—with matching Earth controls—in modified artificial urine medium (mAUMg-high Pi) or LB Lennox supplemented with KNO3, and its formation of biofilms on six different materials was assessed. After one, two, and three days of incubation, the ISS crew terminated subsets of the experiment by fixation in paraformaldehyde, and aliquots of the supernatant were used for the planktonic cell size study presented here. The measurements were obtained post-flight through the use of phase contrast microscopy under oil immersion, a Moticam 10+ digital camera, and the FIJI image analysis program. Statistical comparisons were conducted to identify which treatments caused significant differences in cell dimensions using the Kruskal–Wallis and Dunn tests. There were statistically significant differences as a function of material present in the culture in both LBK and mAUMg-high Pi. Along with this, the data were also grouped by gravitational condition, media, and days of incubation. Comparison of planktonic cells cultured in microgravity showed reduced cell length (from 4% to 10% depending on the material) and diameter (from 1% to 10% depending on the material) with respect to their matching Earth controls, with the caveat that the cultures may have been at different points in their growth curve at a given time. In conclusion, smaller cells were observed on the cultures grown in microgravity, and cell size changed as a function of incubation time and the material upon which the culture grew. We describe these changes here and possible implications for human space travel in terms of crew health and potential applications.

Abstract Many studies have described the response of the facultative anaerobe, Escherichia coli, to anaerobic conditions, yet they all investigated free-living (planktonic) cells because attempts to cultivate anaerobic E. coli biofilm were mostly unsuccessful. We challenged these findings and cultivated E. coli strain MG1655 biofilm under both aerobic and anaerobic conditions, characterizing the mature biofilm architecture and global gene expression profile. We used RNA sequencing technology to compare stationary phase planktonic cells with mature biofilm, cultured with and without oxygen. Our results suggest that gene expression patterns significantly differ between biofilm and planktonic cultures cultivated under the same oxygenic conditions. The anaerobic E. coli biofilms were slow growing and patchy compared to aerobic biofilms, yet some features were unchanged like the production of extracellular polymeric substances. A closer inspection of the mRNA data revealed that essential cell processes were attenuated in anaerobic biofilms, including protein synthesis, information transfer, cell structure, regulation and transport. Our results suggest that lack of oxygen imposes severe stress on mature biofilms thus limiting the cells’ activity. We further propose that E. coli does not favor growing in anaerobic biofilms and when forced to do so, the cells prevail by attenuating their activity in order to survive. This study explores global regulation of Escherichia coli biofilm cultured with and without oxygen.

Risk for infections from Legionella pneumophila for immunocompromised individuals increases greatly when this species is present within the biofilm of the water distribution systems of hospitals or other health facilities. Multiplication and persistence of Legionella may dependent also upon planktonic growth in alternative to sessile growth. Here we compared the persistence of L. pneumophila serogroup 1 in experimental planktonic co-cultures subsided with iron, Pseudomonas aeruginosa and other non Legionella bacteria (quantified as Heterotrophic Plate Count, HPC at 37°C), isolated from drinking water sources of a large hospital. Concentrations of L. pneumophila showed a decreasing pattern with incubation time in all co-cultures, the degree of reduction depending on the experimental treatment. In co-cultures with added P. aeruginosa, no L. pneumophila was detectable already after 4 days of incubation. In contrast in co-cultures without P. aeruginosa, HPC but not iron were significant factors in explaining the pattern of L. pneumophila, although the HPC effect was different according to the incubation time (HPC x time interaction, p < 0.01). Our results highlight the need of controlling for both HPC and metal constituents of the water systems of buildings used by individuals at particular risk to the effects of Legionella exposure.

Planktonic bacteria passing to a sessile state during the formation of a biofilm undergo many gene expression and phenotypic changes. These transformations require a significant time to establish. Inversely, cells extracted from a biofilm should also require a significant time before acquiring the same physiological characteristics as planktonic cells. Relatively few studies have addressed the kinetics of this inverse transformation process. We tested one aspect, namely, the contamination potential of freshly extracted Escherichia coli biofilm cells, precultured in a synthetic medium, in a rich liquid growth medium. We compared the time between inoculation and the beginning of the growth phase of freshly extracted biofilm cells, and suspended exponential and suspended stationary phase cells precultured in the same synthetic medium. Unexpectedly, the lag time for the extracted biofilm cells was the same as the lag time of the suspended exponential phase cells and significantly less than the lag time of the suspended stationary phase cells. The lag times were determined by an impedance technique. Cells extracted from biofilms, i.e., biofilms formed in canalizations and broken up by hydrodynamic forces, are an important source of contamination. Our work shows, in the case of E. coli, the high potential of freshly extracted biofilm cells to reinfect a new medium.