Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978

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.

BackgroundAlthough the most widely accepted mechanism of action for polymyxins is related to bacterial lysis via disruption, we hypothesized that this antimicrobial drug class could have other effects on Pseudomonas aeruginosa planktonic and sessile cells. Little is known regarding oxidative burst and zeta potential (ZP) data associated with the interaction between polymyxin B and P. aeruginosa cells. The present study evaluated endogenous reactive oxygen species (ROS) production and changes in the net charges of biofilm and planktonic cells in response to polymyxin B.ResultsPolymyxin B induced concentration-dependent killing at all concentrations tested in planktonic and sessile cells from P. aeruginosa strains. Sublethal concentrations of polymyxin B induced oxidative burst. ROS production was higher in resistant planktonic cells than in biofilm cells but this was not observed for susceptible cells. Moreover, no net surface charge alterations were observed in planktonic cells from a susceptible strain treated with polymyxin B, but a significant increase of ZP was noted in planktonic cells from a resistant strain.ConclusionOxidative burst generated by planktonic and sessile cells from P. aeruginosa strains against polymyxin B indicates that ROS may have an important role in the mechanism of action of this drug. ZP data revealed that electrostatic interactions of the cationic peptide with the anionic surface of the cells are strain-dependent. Therefore, we suggested that the intracellular effects of polymyxin B should be further investigated to understand polymyxin B-induced stress in P. aeruginosa.

Salmonella enterica forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation has been used to inactivate Salmonella on a variety of foods and contact surfaces, but the relative efficacy of the process against biofilm-associated cells versus free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm-associated cells was determined for three food-borne-illness-associated isolates of Salmonella. Biofilms were formed on sterile glass slides in a coincubation apparatus, using inoculated tryptic soy broth, incubated at 37 degrees C for 48 h. Resulting biofilms were 18 to 24 microm in height as determined by confocal scanning laser microscopy. The planktonic and biofilm cultures were gamma irradiated to doses of 0.0 (control), 0.5, 1.0, 1.5, 2.0 and 2.5 kGy. The D(10) value (the dose of radiation required to reduce a population by 1 log(10), or 90%) was calculated for each isolate-culture based on surviving populations at each radiation dose. The D(10) values of S. enterica serovar Anatum were not significantly (P < 0.05) different for biofilm-associated (0.645 kGy) and planktonic (0.677 kGy) cells. In contrast, the biofilm-associated cells of S. enterica serovar Stanley were significantly more sensitive to ionizing radiation than the respective planktonic cells, with D(10) values of 0.531 and 0.591 kGy, respectively. D(10) values of S. enterica serovar Enteritidis were similarly reduced for biofilm-associated (0.436 kGy) versus planktonic (0.535 kGy) cells. The antimicrobial efficacy of ionizing radiation is therefore preserved or enhanced in treatment of biofilm-associated bacteria.

Risks for development of local and/or systemic infections are the most important complications of catheters that are widely used during hospitalization process. The aims of this study were to investigate and compare the antibiotic susceptibilities of methicillin-resistant staphylococci isolated from catheters, in planktonic and biofilm forms, and to evaluate the antimicrobial effects of antibiotics on those forms alone and in combinations. A total of 30 strains [15 methicillin-resistant Staphylococcus aureus (MRSA) and 15 methicillin-resistant coagulase-negative staphylococci (MR-CNS)] isolated from catheter cultures of patients hospitalized in different clinics and intensive care units in Baskent University Medical School Hospital between 2006-2009, were included in the study. The antibiotic sensitivities of MRSA and MR-CNS isolates were investigated in vitro in planktonic phase and on sessile cells after biofilm was formed. Vancomycin, ciprofloxacin, rifampicin, gentamicin, meropenem, tigecycline, linezolid, ceftazidime and cephazolin were used for antibiotic susceptibility testing. The sensitivity of planktonic cells to antibiotics was primarily investigated, so that minimal inhibitor concentration (MIC) and minimal bactericidal concentration (MBC) values were determined by broth microdilution method. Afterwards, each strain was transformed to sessile cell in a biofilm environment, and MIC and MBC values were also determined for sessile cells. Double and triple antibiotic combinations were prepared, the effectiveness of combinations were studied on both planktonic and biofilm cells with multiple-combination bactericidal testing (MCBT) method. The data set obtained from planktonic and biofilm cells for each antibiotic analyzed via two proportion z test. Statistically significant decreases were found in the sensitivities of sessile cells when compared to planktonic cells (p< 0.01). The tests performed with the use of double and triple antibiotic combinations also showed the susceptibility decrease between planktonic and biofilm forms to be significant in most of the combinations (p< 0.01). The comparison of double and triple antibiotic combinations against planktonic and sessile cells as determined by the inhibition of more than 90% of the strains, revealed no significant difference . Vancomycin and tigecycline were the most effective antibiotics for all isolates in planktonic and sessile cells. Combinations containing vancomycin and rifampicin showed the best activity both double and triple antibiotic combinations against biofilm. In conclusion, our data indicated that combination therapy, especially double combinations of antibiotics seem to be a rational approach for biofilm-related infections.

The human pathogen Listeria monocytogenes forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation effectively inactivates planktonic Listeria, but no information is available on the relative efficacy of the process against biofilm-associated Listeria. The irradiation sensitivity of planktonic or biofilm cells was determined for L. monocytogenes ATCC 43256 and ATCC 49594 and a commonly used surrogate Listeria innocua ATCC 51742. Biofilms were formed on sterile glass slides incubated for 48 h at 22°C, 28°C, or 37°C. The cultures were gamma irradiated and the irradiation D 10 value was calculated for each combination of isolate/culture/temperature. The effect of temperature of cultivation on the irradiation sensitivity of both planktonic cells and biofilm cells varied for each of the isolates. Depending on isolate and temperature, biofilm cells were equally sensitive or more sensitive (P < 0.05) to irradiation. D 10 values overall tended to increase with temperature of cultivation for L. monocytogenes 49594 and L. innocua 51742, but tended to decrease with increasing temperature for L. monocytogenes 43256. The D 10 values of the various culture/temperature combinations differed significantly among the isolates examined. Irradiation effectively eliminates both planktonic and biofilm-associated cells. The extent to which the biofilm habitat modifies the antimicrobial efficacy of irradiation is dependent on the specific isolate examined and the temperature at which it forms. This study is the first inquiry to show that biofilm Listeria cells are as sensitive or more sensitive to irradiation compared with planktonic cells and that this response is dependent on biofilm formation conditions.

Microbially influenced corrosion (MIC) is a process whereby microbial cells living in an exopolymer matrix, known as a biofilm, induce corrosion of the associated surface. The risk of MIC within a system is usually assessed by measuring planktonic (free-floating) cells, which is assumed to approximate the biofilm-associated cells (sessile) cells. This work aimed to determine the accuracy of this industry-practiced approach of assessing MIC risk from planktonic cell counts. Planktonic and sessile cell counts of two single species cultures, one of an aerobe (Pseudomonas fluorescens) and one of an anaerobe (Geoalkalibacter subterraneus), both of which have been associated with MIC, were monitored in a growth curve test to determine how planktonic and sessile cell counts relate during the initial stages of biofilm formation. The results indicate two factors govern biofilm initiation of the tested species. Firstly, a minimum planktonic cell density is required and secondly, a minimum exposure time of the surface are both required prior to biofilm initiation and the onset of internal corrosion of carbon-steel pipelines. Both these factors affect biofilm formation in a species specific manner.

Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

This study aimed to address the significant problems of bacterial biofilms found in medical fields and many industries. It explores the potential of classic photoactive carbon dots (CDots), with 2,2′-(ethylenedioxy)bis (ethylamine) (EDA) for dot surface functionalization (thus, EDA-CDots) for their inhibitory effect on B. subtilis biofilm formation and the inactivation of B. subtilis cells within established biofilm. The EDA-CDots were synthesized by chemical functionalization of selected small carbon nanoparticles with EDA molecules in amidation reactions. The inhibitory efficacy of CDots with visible light against biofilm formation was dependent significantly on the time point when CDots were added; the earlier the CDots were added, the better the inhibitory effect on the biofilm formation. The evaluation of antibacterial action of light-activated EDA-CDots against planktonic B. subtilis cells versus the cells in biofilm indicate that CDots are highly effective for inactivating planktonic cells but barely inactivate cells in established biofilms. However, when coupling with chelating agents (e.g., EDTA) to target the biofilm architecture by breaking or weakening the EPS protection, much enhanced photoinactivation of biofilm-associated cells by CDots was achieved. The study demonstrates the potential of CDots to prevent the initiation of biofilm formation and to inhibit biofilm growth at an early stage. Strategic combination treatment could enhance the effectiveness of photoinactivation by CDots to biofilm-associated cells.

Salmonella has emerged as a well-recognized food-borne pathogen, with many strains able to form biofilms and thus cause cross-contamination in food processing environments where acid-based disinfectants are widely encountered. In the present study, RNA sequencing was employed to establish complete transcriptome profiles of Salmonella Enteritidis in the forms of planktonic and biofilm-associated cells cultured in Tryptic Soytone Broth (TSB) and acidic TSB (aTSB). The gene expression patterns of S. Enteritidis significantly differed between biofilm-associated and planktonic cells cultivated under the same conditions. The assembled transcriptome of S. Enteritidis in this study contained 5,442 assembled transcripts, including 3,877 differentially expressed genes (DEGs) identified in biofilm and planktonic cells. These DEGs were enriched in terms such as regulation of biological process, metabolic process, macromolecular complex, binding and transferase activity, which may play crucial roles in the biofilm formation of S. Enteritidis cultivated in aTSB. Three significant pathways were observed to be enriched under acidic conditions: bacterial chemotaxis, porphyrin-chlorophyll metabolism and sulfur metabolism. In addition, 15 differentially expressed novel non-coding small RNAs (sRNAs) were identified, and only one was found to be up-regulated in mature biofilms. This preliminary study of the S. Enteritidis transcriptome serves as a basis for future investigations examining the complex network systems that regulate Salmonella biofilm in acidic environments, which provide information on biofilm formation and acid stress interaction that may facilitate the development of novel disinfection procedures in the food processing industry.

Candida glabrata has emerged as a common cause of serious life-threatening fungal infections, largely owing to their low susceptibility to azole antifungals. Recent guidance indicates the use of echinocandins as the first-choice drug for the treatment of systemic infections of C. glabrata; however, C. glabrata resistance to echinocandins is reportedly increasing. Herein, we present the induction of anidulafungin resistance in planktonic and sessile cells of C. glabrata and the development of fluconazole crossresistance. MICs of 21 clinical C. glabrata strains were determined by a broth microdilution method using anidulafungin and fluconazole. Biofilm formation on a tracheal catheter was determined using 1- × 1-cm2 polyvinyl polychloride catheter fragments. Induction of anidulafungin resistance in planktonic and sessile cells and evaluation of its stability were performed by exposing the strains to successively higher concentrations of the antifungal. The induction resulted in strains strongly resistant to anidulafungin (MICs: 1−2 μg/mL) and fluconazole (≥64 μg/mL). Most of the sessile cells of C. glabrata presented slightly reduced susceptibility compared with the planktonic cells. Clinically, this cross-resistance could lead to therapeutic failure while using fluconazole in patients previously exposed to subinhibitory concentrations of anidulafungin for extended periods.

Visualization of initial attachment of bioleaching bacteria using combined atomic force and epifluorescence microscopy

IntroductionCryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity.MethodsIn the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans.ResultsBTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 μg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 μg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties.ConclusionThese results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.

During biofilm growth, the coexistence of planktonic and sessile cells can lead to dynamic exchanges between the two populations. We have monitored the fate of these populations in glass tube assays, where the Bacillus thuringiensis 407 strain produces a floating pellicle. Time-lapse spectrophotometric measurement methods revealed that the planktonic population grew until the pellicle started to be produced. Thereafter, the planktonic population decreased rapidly down to a value close to zero while the biofilm was in continuous growth, showing no dispersal until 120 h of culture. We found that this decrease was induced by the presence of the pellicle, but did not occur when oxygen availability was limited, suggesting that it was independent of cell death or cell sedimentation and that the entire planktonic population has integrated the biofilm. To follow the distribution of recruited planktonic cells within the pellicle, we tagged planktonic cells with GFP and sessile cells with mCherry. Fluorescence binocular microscopy observations revealed that planktonic cells, injected through a 24-h-aged pellicle, were found only in specific areas of the biofilm, where the density of sessile cells was low, showing that spatial heterogeneity can occur between recruited cells and sessile cells in a monospecies biofilm.

Effects of oregano essential oil and carvacrol on biofilms of Staphylococcus aureus from food-contact surfaces

Ionizing radiation effectively inactivates Escherichia coli O157:H7, but the efficacy of the process against biofilm cells versus that against free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm cells was determined for three isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894). Biofilms were formed on sterile glass slides incubated at 37 degrees C for either 24 h, 48 h, or 72 h. The biofilm and planktonic cultures were gamma irradiated at doses ranging from 0.0 (control) to 1.5 kGy. The dose of radiation value required to reduce the population by 90% (D10) was calculated for each isolate, culture, and maturity based on viable populations at each radiation dose. For each of the times sampled, the D10 values of isolate 43894 planktonic cells (0.454 to 0.479 kGy) were significantly (P<0.05) higher than those observed for biofilm cells (0.381 to 0.385 kGy), indicating a significantly increased sensitivity to irradiation for cells in the biofilm habitat. At the 24-h sampling time, isolate C9490 showed a similar pattern, in which the D10 values of planktonic cells (0.653 kGy) were significantly higher than those for biofilm cells (0.479 kGy), while isolate 35150 showed the reverse, with D10 values of planktonic cells (0.396 kGy) significantly lower than those for biofilm cells (0.526 kGy). At the 48-h and 72-h sampling times, there were no differences in radiation sensitivities based on biofilm habitat for C9490 or 35150. Biofilm-associated cells, therefore, show a response to irradiation which can differ from that of planktonic counterparts, depending on the isolate and the culture maturity. Culture maturity had a more significant influence on the irradiation efficacy of planktonic cells but not on biofilm-associated cells of E. coli O157:H7.