Effect of combination of doxycycline with natural products against planktonic cells, biofilm, and virulence factor of Pseudomonas aeruginosa

Biofilm formations play an important role in Staphylococcus aureus pathogenesis and contribute to antibiotic treatment failures in biofilm-associated infections. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profiles of cefquinome against an experimental catheter-related biofilm model due to S. aureus, including three clinical isolates and one non-clinical isolate. The minimal inhibitory concentration (MIC), minimal biofilm inhibitory concentration (MBIC), biofilm bactericidal concentration (BBC), minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) and in vitro time-kill curves of cefquinome were studied in both planktonic and biofilm cells of study S. aureus strains. The in vivo post-antibiotic effects (PAEs), PK profiles and efficacy of cefquinome were performed in the catheter-related biofilm infection model in murine. A sigmoid Emax model was utilized to determine the PK/PD index that best described the dose-response profiles in the model. The MICs and MBICs of cefquinome for the four S. aureus strains were 0.5 and 16 μg/mL, respectively. The BBCs (32–64 μg/mL) and MBECs (64–256 μg/mL) of these study strains were much higher than their corresponding BPC values (1–2 μg/mL). Cefquinome showed time-dependent killing both on planktonic and biofilm cells, but produced much shorter PAEs in biofilm infections. The best-correlated PK/PD parameters of cefquinome for planktonic and biofilm cells were the duration of time that the free drug level exceeded the MIC (fT > MIC, R2 = 96.2%) and the MBIC (fT > MBIC, R2 = 94.7%), respectively. In addition, the AUC24h/MBIC of cefquinome also significantly correlated with the anti-biofilm outcome in this model (R2 = 93.1%). The values of AUC24h/MBIC for biofilm-static and 1-log10-unit biofilm-cidal activity were 22.8 and 35.6 h; respectively. These results indicate that the PK/PD profiles of cefquinome could be used as valuable guidance for effective dosing regimens treating S. aureus biofilm-related infections.

Biofilms related to human infection have high levels of pathogenicity due to their resistance to antimicrobial agents. The discovery of antibiofilm agents is necessary. One approach to overcome this problem is the use of antibiotics agents' combination. This study aimed to determine the efficacy of the combination of natural products thymol and piperine with three aminoglycosides antibiotics, amikacin, kanamycin, and streptomycin against biofilm-forming Salmonella enterica. The microtiter plate assay method was used to evaluate the biofilm-producing capacity of the isolates. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined by the broth microdilution method. The inhibition of biofilm formation and biofilm eradication was determined using the microtiter broth method. The checkerboard method was used to determine the combined effects of natural products with aminoglycosides antibiotics. All the tested isolates showed various levels of biofilm formation. Overall, combinations provided 43.3% of synergy in preventing the biofilm formation and 40% of synergy in eradicating preformed biofilms, and in both cases, no antagonism was observed. The combination of thymol with kanamycin showed a synergistic effect with 16- to 32-fold decrease of the minimum biofilm eradication concentration (MBEC) of kanamycin. The interaction of piperine with amikacin and streptomycin also revealed a synergistic effect with 16-fold reduction of the minimum biofilm inhibitory concentration (MBIC). The combination of thymol with the three antibiotics showed a strong synergistic effect in both inhibiting the biofilm formation and eradicating the preformed biofilm. This study demonstrates that thymol and piperine potentiate the antibiofilm activity of amikacin, kanamycin, and streptomycin. These combinations are a promising approach therapeutic to overcome the problem of Salmonella enterica biofilm-associated infections. In addition, these combinations could help reduce the concentration of individual components, thereby minimizing the nephrotoxicity of aminoglycosides antibiotics.

Susceptibility testing of bacteria to disinfecting chemical agents isolated from dental unit waterlines (DUWL) is necessary for the development of effective disinfectant products. However, until now, susceptibility tests for chemical agents, which are components of DUWL disinfectant products, have not been conducted on bacteria isolated from DUWL water. The aim of this study was to evaluate and compare the susceptibilities of DUWL isolates in planktonic and biofilm states to cetylpyridinium chloride, as well as to the four chemical agents currently used for DUWL management. A total of 56 isolates, including 12 genera, were identified by 16S rDNA sequencing, and one strain of each genus was selected for susceptibility testing. A total of 12 isolates were used for the susceptibility tests. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the planktonic state and the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) for the biofilm state using microtiter plates. MIC, MBC, MBIC, and MBEC of the 12 isolates for ethanol were the highest, followed by sodium hypochlorite, hydrogen peroxide, and chlorhexidine. Similar to chlorhexidine, the lowest MIC, MBC, MBIC, and MBEC were found in cetylpyridinium chloride. The susceptibilities of the isolates for sodium hypochlorite and ethanol were similar in the planktonic and biofilm states. For hydrogen peroxide and chlorhexidine, the MBIC and MIC were similar, but MBEC was 256 times higher than MBC. The MBIC and MBEC of isolates for cetylpyridinium chloride were 128 and 256 times higher than the MIC and MBC, respectively. As far as we know, this was the first study reporting the susceptibility of DUWL isolates to cetylpyridinium chloride and chemical agents used for disinfecting DUWLs. Cetylpyridinium chloride, for which the DUWL isolates showed the highest susceptibility, could be used for disinfecting DUWLs.

Diabetic foot ulcers (DFUs) are the main cause of hospitalizations and amputations in diabetic patients. Failure of standard foot care is the most important cause of impaired DFU healing. Dakin's solution (DS) is a promising broad-spectrum bactericidal antiseptic for management of DFUs. Studies investigating the efficacy of using DS on the healing process of DFUs are scarce. Accordingly, this is the first evidence-based, randomized, controlled trial conducted to evaluate the effect of using diluted DS compared with the standard care in the management of infected DFUs. A randomized controlled trial was conducted to assess the efficacy of DS in the management of infected DFUs. Patients were distributed randomly to the control group (DFUs irrigated with normal saline) or the intervention group (DFUs irrigated with 0.1% DS). Patients were followed for at least 24 weeks for healing, reinfection, or amputations. In vitro antimicrobial testing on DS was performed, including determination of its minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration, minimum biofilm eradication concentration, and suspension test. Replacing normal saline irrigation in DFU standard care with 0.1% DS followed by soaking the ulcer with commercial sodium hypochlorite (0.08%) after patient discharge significantly improved ulcer healing (P < .001) and decreased the number of amputations and hospitalizations (P < .001). The endpoint of death from any cause (risk ratio, 0.13; P = .029) and the amputation rate (risk ratio, 0.27; P < .001) were also significantly reduced. The effect on ulcer closure (OR, 11.9; P < .001) was significantly enhanced in comparison with the control group. Moreover, DS irrigation for inpatients significantly decreased bacterial load (P < .001). The highest values for the in-vitro analysis of DS were as follows: minimum inhibitory concentration (MIC), 1.44%; minimum bactericidal concentration (MBC), 1.44%; minimum biofilm inhibitory concentration (MBIC), 2.16%; and minimum biofilm eradication concentration (MBEC), 2.87%. Compared with standard care, diluted DS (0.1%) was more effective in the management of infected DFUs. Dakin's solution (0.1%) irrigation with debridement followed by standard care is a promising method in the management of infected DFUs.

The aim of this study was to investigate the susceptibility of Mutans streptococci in both planktonic and biofilm states to erythrosine.S. mutans was cultured in brain-heart infusion (BHI) broth.Erythrosine was diluted in BHI broth and prepared at a concentration range of 0.02 -10000 g/L.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured using the microdilution method.After forming biofilms on 96-well plates, the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were measured.S. mutans was susceptible to erythrosine in both planktonic and biofilm states.MIC and MBC values were both 19.5 g/L for the planktonic state, while MBIC and MBEC values were 313 g/L and 2500 g/L, respectively, for the biofilm state.Erythrosine (19.5 g/L) exhibited a bactericidal effect on S. mutans (killing 99.9%) in the planktonic state.For biofilms, erythrosine inhibited biofilm growth and eradicated 99.9% of biofilm bacteria at higher concentrations than MIC and MBC.These MBIC and MBEC concentrations are much lower than known noxious doses, and the MIC, MBC, and MBIC values were even lower than clinical concentrations.

With the spread of multi-drug-resistant (MDR) bacteria and the lack of effective antibiotics to treat them, developing new therapeutic methods and strategies is essential. In this study, we evaluated the antibacterial and antibiofilm activity of different formulations composed of ibuprofen (IBP), acetylsalicylic acid (ASA), and dexamethasone sodium phosphate (DXP) in combination with ciprofloxacin (CIP), gentamicin (GEN), cefepime (FEP), imipenem (IPM), and meropenem (MEM) on clinical isolates of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) as well as the transcription levels of biofilm-associated genes in the presence of sub-MICs of IBP, ASA, and DXP. The minimal inhibitory concentrations (MICs), minimal biofilm inhibitory concentrations (MBICs), and minimum biofilm eradication concentrations (MBECs) of CIP, GEN, FEP, IPM, and MEM with/without sub-MICs of IBP (200 µg/mL), ASA (200 µg/mL), and DXP (500 µg/mL) for the clinical isolates were determined by the microbroth dilution method. Quantitative real-time-PCR (qPCR) was used to determine the expression levels of biofilm-related genes, including icaA in S. aureus and algD in P. aeruginosa at sub-MICs of IBP, ASA, and DXP. All S. aureus isolates were methicillin-resistant S. aureus (MRSA), and all P. aeruginosa were resistant to carbapenems. IBP decreased the levels of MIC, MBIC, and MBEC for all antibiotic agents in both clinical isolates, except for FEP among P. aeruginosa isolates. In MRSA isolates, ASA decreased the MICs of GEN, FEP, and IPM and the MBICs of IPM and MEM. In P. aeruginosa, ASA decreased the MICs of FEP, IPM, and MEM, the MBICs of FEP and MEM, and the MBEC of FEP. DXP increased the MICs of CIP, GEN, and FEP, and the MBICs of CIP, GEN, and FEP among both clinical isolates. The MBECs of CIP and FEP for MRSA isolates and the MBECs of CIP, GEN, and MEM among P. aeruginosa isolates increased in the presence of DXP. IBP and ASA at 200 µg/mL significantly decreased the transcription level of algD in P. aeruginosa, and IBP significantly decreased the transcription level of icaA in S. aureus. DXP at 500 µg/mL significantly increased the expression levels of algD and icaA genes in S. aureus and P. aeruginosa isolates, respectively. Our findings showed that the formulations containing ASA and IBP have significant effects on decreasing the MIC, MBIC, and MBEC levels of some antibiotics and can down-regulate the expression of biofilm-related genes such as icaA and algD. Therefore, NSAIDs represent appropriate candidates for the design of new antibacterial and antibiofilm therapeutic formulations.

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.

BackgroundAcinetobacter baumannii is an important pathogen in clinical infections, and biofilm formation is an effective way for A. baumannii to survive under external pressures. In this study, the aims were to examine the antimicrobial resistance, biofilm formation, and biofilm-specific resistance in clinical isolates of A. baumannii.Materials and MethodsA total of 104 clinical A. baumannii isolates were collected from a large teaching hospital in Southwest China. The antibiotics susceptibilities were tested, and biofilm-forming ability was evaluated by crystal violet staining by confocal laser scanning microscopy (CLSM). Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) of ciprofloxacin, meropenem, and ceftazidime were tested on selected strains by broth microdilution method. Biofilm-associated genes were detected by polymerase chain reaction (PCR), and expression of genes at planktonic stage and biofilm stage were analyzed by real-time reverse transcription PCR (RT-PCR).ResultsMultidrug-resistant (MDR) isolates accounted for 65.4%, but no strain was resistant to tigecycline and polymyxin B. Moreover, non-MDR strains tended to form stronger biofilms than MDR strains, and a negative correlation between biofilm-forming ability and resistance profiles to each of tested antimicrobials were observed. The MBECs and MBICs of ciprofloxacin, ceftazidime, and meropenem were evidently increased compared with MICs and MBCs among all tested strains. Additionally, the biofilm formation ability of the csuD-positive strains was stronger than that of the csuD-negative strains. The strains in MDR group had higher carrying rate of csuA and csuD genes than non-MDR group, while non-MDR strains possessed more ompA gene than MDR group. Finally, abaI gene was significantly up-regulated after biofilm formation.ConclusionThese results revealed valuable data for the negative correlation between antimicrobial resistance and biofilm formation, as well as phenotypic and genotypic characteristics of biofilm formation in A. baumannii.

Although the most common bacteria in the supragingival plaque are Gram-positive streptococci, no extensive investigations have been conducted into the susceptibility of these species to chlorhexidine and cetylpyridinium chloride. Therefore, in this study, we investigated the susceptibility of 80 streptococcal strains in planktonic or biofilm states to these two antimicrobial agents. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the planktonic streptococci were measured using the microdilution method, as were the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) measured on streptococcal biofilms formed on 96-well plates. In all species, the MIC, MBC, MBIC, and MBEC values were higher for chlorhexidine than for cetylpyridinium chloride, with sensitivity values varying according to species. For chlorhexidine, the MIC, MBC, and MBIC values showed statistically significant differences among species. However, only MBEC values showed statistically significant differences for cetylpyridinium chloride. The MIC against Streptococcus mutans and the MBC against Streptococcus salivarius were significantly lower than those against the other species. With he exception of a few species, most of the bacterium susceptibility values were higher in the biofilm state than in the planktonic state.

Biofilm, naturally formed by microorganisms as integrated surface-bound communities, is one of the reasons for the development of antimicrobial resistance. Haemophilus spp. are common and representative opportunistic Gram-negative rods forming from the upper respiratory tract microbiota. The aim of this paper was to evaluate the influence of thiazolidine-2,4-dionebased azolidine and chlorophenylthiosemicarbazone hybrids against both planktonic and biofilm-forming Haemophilus spp. cells. The in vitro activity against planktonic and biofilm-forming cells of the tested compounds were evaluated by using the broth microdilution method. These activities were detected against reference and clinical strains of Haemophilus spp. on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The target compounds showed potential activity against planktonic cells with MIC = 62.5–500 mg/L and biofilm-forming cells with MBIC = 62.5–1000 mg/L. The observed anti-adhesive properties of the tested compounds were reversible during long-term incubation in a lower concentration of compounds.

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.

Biofilms cause chronic infections that are difficult to treat because of the resistance of microorganisms inside them. The goal is to assess the killing potential of cefotaxime against Escherichia coli in biofilms obtained from clinical isolates kept in the clinical microbiology laboratory of Dr. Soetomo Surabaya Hospital. The study was conducted by laboratory experimental design. Two steps challenged the bacterial isolates by applying cefotaxime to the planktonic bacterial state and the biofilm state. Minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration (MBIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) were used as an indicator for comparison. Nineteen isolates of E coli were used for this experiment, 7 isolates with MIC of 0.125-0.25 μg/ml (36,84%), 6 isolates with MBC value 0.25-0.5 μg/ml (31,57%). The MBIC &gt; 128 μg/ml is 6 isolates (31,57%), MBEC &gt; 128 μg/ml is 14 isolates (73,68%). Cefotaxime had lower killing efficacy against E. coli in biofilm than in the planktonic phase. MBIC of E. coli requires cefotaxime at minimal 5 times two-fold increase from MIC, with an average 7-8 times. MBEC of E. coli requires cefotaxime concentration minimal 5 times two-fold dilution increase from MBC, with an average 8 - 9 times.&#x0D;

In this study, a biodegradable poly-gamma-glutamic-acid nanopolymer (Ɣ-PGA NP) was investigated for its activity against clinical strains of Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli), and reference strains of S. aureus ATCC 6538, S. pyogenes ATCC 19615 (Gram-positive), and Gram-negative E. coli ATCC 25922, and K. pneumoniae ATCC 13884 bacterial biofilms. The minimum inhibitory concentration (MIC) effect of Ɣ-PGA NP showed inhibitory effects of 0.2, 0.4, 1.6, and 3.2 μg/mL for S. pyogenes, S. aureus, E. coli, and K. pneumoniae, respectively. Also, MIC values were 1.6, 0.8, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Afterwards, MBEC (minimum biofilm eradication concentration) and MBIC (minimum biofilm inhibitory concentration) were investigated to detect Ɣ-PGA NPs efficiency against the biofilms. MBEC and MBIC increased with increasing Ɣ-PGA NPs concentration or time of exposure. Interestingly, MBIC values were at lower concentrations of Ɣ-PGA NPs than those of MBEC. Moreover, MBEC values showed that K. pneumoniae was more resistant to Ɣ-PGA NPs than E. coli, S. aureus, and S. pyogenes, and the same pattern was observed in the reference strains. The most effective results for MBEC were after 48 h, which were 1.6, 0.8, 0.4, and 0.2 µg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. Moreover, MBIC results were the most impactful after 24 h but some were the same after 48 h. MBIC values after 48 h were 0.2, 0.2, 0.2, and 0.1 μg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. The most effective results for MBEC were after 24 h, which were 1.6, 0.8, 0.4, and 0.4 µg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Also, MBIC results were the most impactful after an exposure time of 12 h. MBIC values after exposure time of 12 h were 0.4, 0.4, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Besides that, results were confirmed using confocal laser scanning microscopy (CLSM), which showed a decrease in the number of living cells to 80% and 60% for MBEC and MBIC, respectively, for all the clinical bacterial strains. Moreover, living bacterial cells decreased to 70% at MBEC while decreasing up to 50% at MBIC with all bacterial refence strains. These data justify the CFU quantification. After that, ImageJ software was used to count the attached cells after incubating with the NPs, which proved the variation in live cell count between the manual counting and image analysis methods. Also, a scanning electron microscope (SEM) was used to detect the biofilm architecture after incubation with the Ɣ-PGA NP. In in vivo wound healing experiments, treated wounds of mice showed faster healing (p < 0.00001) than both the untreated mice and those that were only wounded, as the bacterial count was eradicated. Briefly, the infected mice were treated faster (p < 0.0001) when infected with S. pyogenes > S. aureus > E. coli > K. pneumoniae. The same pattern was observed for mice infected with the reference strains. Wound lengths after 2 h showed slightly healing (p < 0.001) for the clinical strains, while treatment became more obvious after 72 h > 48 h > 24 h (p < 0.0001) as wounds began to heal after 24 h up to 72 h. For reference strains, wound lengths after 2 h started to heal up to 72 h.

This study compares the effect of heavy metals (Hg2+, Cu2+, and Pb2+) on the Rhodotorula mucilaginosa and Saccharomyces boulardii biofilm and planktonic cells. A MBECTM-HTP assay was used to test the levels of tolerance to heavy metals. The minimum inhibitory concentration (MICp) and minimum lethal concentration (MLCp) of the R. mucilaginosa and S. boulardii planktonic cells were determined, as well as minimum biofilm eradication concentration (MBEC). Metal removal efficiency was determined by batch biosorption assay. Previous studies had focused on heavy metal tolerance and removal efficiency of planktonic cells from Rhodotorula species only. Hence, our study presents and compares results for metal tolerance and removal efficiency of the R. mucilaginosa planktonic cells and biofilm. Biofilm tolerance was higher than the planktonic cells. The R. mucilaginosa planktonic cells showed the tolerance in the presence of Hg2+ (MICp 0.08 mM), Cu2+ (MICp 6.40 mM), and Pb2+ (MICp 3.51 mM), while the S. boulardii planktonic cells only tolerated Pb2+ (MICp 0.43 mM). The R. mucilaginosa biofilm showed the highest tolerance in the presence of Hg2+ (MBEC >0.31 mM), Cu2+(MBEC >12.81 mM), Pb2+ (MBEC >7.12 mM), and obtained results were confirmed by fluorescence microscopy. S. boulardii did not show potential in biofilm formation. The R. mucilaginosa biofilm exhibited better efficiency in removal of all tested metals than the planktonic cells. Metal removal efficiency was in the range from 4.79–10.25% for planktonic cells and 91.71–95.39% for biofilm.

The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species. Linezolid was the most effective drug in inhibiting staphylococci in the biofilm, without an increase in the MIC, when compared to planktonic cells. None of the isolates were resistant to this drug.