Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci.

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 pathogenic effect of Staphylococci is due to extra-cellular factors and properties such as adherence and biofilm production. The nature of the biofilm and the physiological properties of biofilm-producing bacteria result in an inherent antibiotic resistance and require further investigation. Two hundred and sixty Staphylococcal strains were cultured from 600 clinical specimens obtained from hospitalized patients. Among these, 155 were identified as coagulase-positive (CPS) and 105 as coagulase-negative (CNS) staphylococci. Staphylococcal strains were tested for biofilm production using the tissue culture plate (TCP) method. TCP detection showed that of the 155 CPS, 124 (80%) were biofilm producers, while 63 (60%) of the 105 CNS were biofilm producers. Biofilm-producing strains were scanned by scanning electron microscope (SEM) to confirm biofilm formation, study biofilm production, and examine antibiotic effects on biofilm formation. Disc diffusion method was used to study resistance of planktonic and biofilm-forming cells to antibiotics. Planktonic cells were less resistant to antibiotics than biofilm-forming cells. Microbroth dilution method and a new BioTimer assay were used to determine antibiotic MICs affecting planktonic and biofilm cells. Both methods showed that the MICs for planktonic cells were less than that for biofilm cells. The BioTimer assay was therefore found to be sensitive, accurate, and reliable, with results in agreement with those from the broth dilution method and SEM.

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.

Background: Pseudomonas aeruginosa is an important environmental, opportunistic, and nosocomial pathogen with a significant threat to public health. Combination therapy has many advantages due to the simultaneous action of two drugs on two separate cellular targets. In the present study, the effect of the combination of doxycycline and natural products against planktonic cells, biofilm, and virulence factors of P. aeruginosa was evaluated. Methods: To perform this work, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of doxycycline and natural products were determined by broth microdilution method. The microtiter plate assay method was used to determine the minimal biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC). The effect of doxycycline and natural products against pyocyanin, swarming motility, and swimming motility was evaluated. The checkerboard method was used to evaluate the effect of the combination of doxycycline with natural products against planktonic and biofilm cells. Results: The MIC of doxycycline ranges between 2 µg/mL and 128 µg/mL with an average of 35.89 µg/mL. Sinapic acid shows the best inhibitory activity against planktonic cells with an average MIC of 27.79 µg/mL. At the sub-inhibitory concentrations, the pyocyanin production, swarming motility, and swimming motility decrease. Out of the six combinations tested, the combination formed by doxycycline and sinapic acid exhibited synergistic activity for the prevention of biofilm formation with a 7-fold reduction in MBIC of doxycycline. Conclusion: This study revealed that the doxycycline and sinapic acid combination could be considered as a promising candidate for the development of therapy against P. aeruginosa infections.

An in vitro overview of the inhibitory effects of selected fluoroquinolones against planktonic and biofilm cells of the methicillin-resistant Staphylococcus aureus (MRSA) strain American type culture collection (ATCC) 43300 and the Pseudomonas aeruginosa strain ATCC 27853 was carried out. Biofilm cells of both strains were less susceptible to the selected antibiotics than their planktonic counterparts. In addition, certain antibiotics were more effective against biofilm cells, while others performed better on the planktonic cells. Against P. aeruginosa, ciprofloxacin was the most potent on both planktonic and biofilm cells, whereas ofloxacin was the least potent on both biofilm and planktonic cells. Moxifloxacin and gatifloxacin were the most potent against both planktonic and biofilm MRSA bacteria, however, not in the same order of activity. Norfloxacin was the least active when tested against both planktonic and biofilm cells. The results of this work are expected to provide insight into the efficacy of various fluoroquinolones against MRSA and Pseudomonas aeruginosa biofilms. This study could form the basis for future clinical studies that could recommend special guidelines for the management of infections that are likely to involve bacteria in their biofilm state.

Fluconazole and biogenic silver nanoparticles-based nano-fungicidal system for highly efficient elimination of multi-drug resistant Candida biofilms

Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis

Diatoms are abundant in biofilms developed on surfaces immersed in sunlit waters. In both the planktonic and the biofilm mode of growth, diatoms produce carbohydrate polymers which perform several functions including motility, protection, production of macro-aggregates and detoxification. However, little is known about the differences, if any, in the production and characterization at the molecular level of carbohydrates in planktonic and biofilm cells. In order to identify the differences in these two modes of growth, the concentration of total carbohydrates, carbohydrate fractions, neutral carbohydrates, uronic acids and amino sugars in planktonic and biofilm cells of Amphora rostrata were measured. The results showed that the distribution of carbohydrate fractions, uronic acids and amino sugars was different in biofilm and planktonic cells. Cell normalized concentrations of these components were two to five times greater in planktonic cells compared with biofilm cells. The concentrations of glucose and glucosamine decreased, whereas fucose increased in planktonic cells over the period of cultivation. Conversely, the concentrations of glucose and glucosamine increased while that of fucose decreased in attached cells. The study suggests that marked differences exist between the carbohydrates of the planktonic and the biofilm cells of A. rostrata.

Biofilm formation by coagulase-negative staphylococci: Impact on the efficacy of antimicrobials and disinfectants commonly used on dairy farms

Formation of viable, but putatively non-culturable (VPNC) cells of beer-spoilage lactobacilli growing in biofilms

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of 14C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, μmax = 10.08 ± 1.2/day; half-saturation constant, KS = 3.98 ± 1.28 mg/L; substrate inhibition constant, KI = 42.78 ± 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate 14C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535–546, 1997.

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, micro(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed.

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

Antimicrobial mechanism of luteolin against Staphylococcus aureus and Listeria monocytogenes and its antibiofilm properties

During this study, we have investigated in vitro activity of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide derivatives with N-ethyl, N-(4-metoxyphenyl) and N-cyclohexyl substituents against Gram-negative Haemophilus influenzae and H. parainfluenzae bacteria. A spectrophotometric assay was used in order to determine the bacterial growth and biofilm formation using a microtiter plate to estimate minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC). Among the tested N-substituted pyrazole derivatives, only N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide showed a significant in vitro activity against both planktonic cells of H. parainfluenzae (MIC = 0.49–31.25 μg ml−1) and H. influenzae (MIC = 0.24–31.25 μg ml−1) as well as biofilm-forming cells of H. parainfluenzae (MBIC = 0.24–31.25 μg ml−1) and H. influenzae (MBIC = 0.49 to ≥31.25 μg ml−1). The pyrazole compound exerted higher inhibitory effect both on the growth of planktonic cells and biofilm formation by penicillinase-positive and penicillinase-negative isolates of H. parainfluenzae than the activity of commonly used antibiotics such as ampicillin. No cytotoxicity of the tested compound in vitro at concentrations used was found. The tested pyrazole N-ethyl derivative could be considered as a compound for the design of agents active against both pathogenic H. influenzae and opportunistic H. parainfluenzae, showing also anti-biofilm activity. This appears important because biofilms are determinants of bacterial persistence in long-term and recurrent infections recalcitrant to standard therapy.