Effects Of Subminimal Inhibitory Concentrations Research Articles

Sub-MIC antibiotics influence the microbiome, resistome and structure of riverine biofilm communities.

The effects of sub-minimum inhibitory concentrations (sub-MICs) of antibiotics on aquatic environments is not yet fully understood. Here, we explore these effects by employing a replicated microcosm system fed with river water where biofilm communities were continuously exposed over an eight-week period to sub-MIC exposure (1/10, 1/50, and 1/100 MIC) to a mix of common antibiotics (ciprofloxacin, streptomycin, and oxytetracycline). Biofilms were examined using a structure-function approach entailing microscopy and metagenomic techniques, revealing details on the microbiome, resistome, virulome, and functional prediction. A comparison of three commonly used microbiome and resistome databases was also performed. Differences in biofilm architecture were observed between sub-MIC antibiotic treatments, with an overall reduction of extracellular polymeric substances and autotroph (algal and cyanobacteria) and protozoan biomass, particularly at the 1/10 sub-MIC condition. While metagenomic analyses demonstrated that microbial diversity was lowest at the sub-MIC 1/10 antibiotic treatment, resistome diversity was highest at sub-MIC 1/50. This study also notes the importance of benchmarking analysis tools and careful selection of reference databases, given the disparity in detected antimicrobial resistance genes (ARGs) identity and abundance across methods. Ultimately, the most detected ARGs in sub-MICs exposed biofilms were those that conferred resistance to aminoglycosides, tetracyclines, β-lactams, sulfonamides, and trimethoprim. Co-occurrence of microbiome and resistome features consistently showed a relationship between Proteobacteria genera and aminoglycoside ARGs. Our results support the hypothesis that constant exposure to sub-MICs antibiotics facilitate the transmission and promote prevalence of antibiotic resistance in riverine biofilms communities, and additionally shift overall microbial community metabolic function.

Anti-Biofilm Potential of Lactobacillus casei and Lactobacillus rhamnosus Cell-Free Supernatant Extracts against Staphylococcus aureus.

Biofilm production is an important virulence factor in Staphylococcus aureus. Most of the infections associated with biofilms of this bacterium are very difficult to treat using antibiotics. The present research studied the effects of the two probiotic Lactobacillus species L. casei and L. rhamnosus on S. aureus biofilm. Cell-free supernatant (CFS) extracts of L. casei ATCC 39392 and L. rhamnosus ATCC 7469 culture were prepared. The effects of sub-minimum inhibitory concentrations of the CFS extracts on cell surface hydrophobicity (CSH), initial attachment, biofilm formation, and their ability in eradicating S. aureus ATCC 33591 biofilms were assessed. In addition, the effects of CFS extracts on expression of the genes involved in formation of S. aureus biofilms (cidA, hld, sarA, icaA, and icaR) were also evaluated through real-time polymerase chain reaction. CFSs of both Lactobacillus spp. significantly reduced CSH, initial attachment, and biofilm formation and eradicated the biofilms. The above findings were supported by scanning electron microscopy results. These two Lactobacillus CFSs significantly changed the expression of all studied biofilm-related genes. Expression levels of cidA, hld, and icaR genes significantly increased by 4.4, 2.3, and 4.76 fold, respectively, but sarA and icaA genes were significantly downregulated by 3.12 and 2.3 fold. The results indicated that CFS extracts of L. casei and L. rhamnosus had desirable antagonistic and anti-biofilm effects against S. aureus. Consequently, carrying out further research enables us to prepare pharmaceuticals from these CFSs in order to prevent and treat infections caused by S. aureus biofilms.

Effects of Sub-Minimum Inhibitory Concentrations of Gentamicin on Alginate Produced by Clinical Isolates of Pseudomonas aeruginosa.

Bacterial virulence factors may be influenced by sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. The main purpose of this study was to investigate the effects of gentamicin at sub-MICs (0.5 MIC and 0.25 MIC) on alginate production of clinical isolates of Pseudomonas aeruginosa. The minimum inhibitory concentrations of gentamicin against 88 clinical isolates of P. aeruginosa were determined using the broth microdilution method. Alginate production of the isolates in the absence and presence of gentamicin at sub-MICs was assessed by the carbazole method. The presence of alginate in clinical isolates was confirmed by the detection of alginate genes (algD and algU) using the PCR method. All the isolates had the ability of alginate production and were positive for algD and algU genes. sub-MICs of gentamicin significantly increased alginate production of 34 isolates (38.6%). On the other hand, in 49 isolates (55.7%), alginate production was significantly increased after treatment with sub-MICs of gentamicin. In five isolates (5.7%), the alginate production was reduced in exposure to 0.5 MIC of gentamicin while it was increased by gentamicin at 0.25 MIC. This study showed different effects of gentamicin at sub-MICs on the alginate production of clinical isolates of P. aeruginosa. Further research is highly recommended to understand the mechanism of different responses of P. aeruginosa isolates to the exposure of sub-MICs of gentamicin.

Effects of Sub-Minimum Inhibitory Concentrations of Imipenem and Colistin on Expression of Biofilm-Specific Antibiotic Resistance and Virulence Genes in Acinetobacter baumannii Sequence Type 1894.

Antibiotics at suboptimal doses promote biofilm formation and the development of antibiotic resistance. The underlying molecular mechanisms, however, were not investigated. Here, we report the effects of sub-minimum inhibitory concentrations (sub-MICs) of imipenem and colistin on genes associated with biofilm formation and biofilm-specific antibiotic resistance in a multidrug-tolerant clinical strain of Acinetobacter baumannii Sequence Type (ST) 1894. Comparative transcriptome analysis was performed in untreated biofilm and biofilm treated with sub-MIC doses of imipenem and colistin. RNA sequencing data showed that 78 and 285 genes were differentially expressed in imipenem and colistin-treated biofilm cells, respectively. Among the differentially expressed genes (DEGs), 48 and 197 genes were upregulated exclusively in imipenem and colistin-treated biofilm cells, respectively. The upregulated genes included those encoding matrix synthesis (pgaB), multidrug efflux pump (novel00738), fimbrial proteins, and homoserine lactone synthase (AbaI). Upregulation of biofilm-associated genes might enhance biofilm formation when treated with sub-MICs of antibiotics. The downregulated genes include those encoding DNA gyrase (novel00171), 30S ribosomal protein S20 (novel00584), and ribosome releasing factor (RRF) were downregulated when the biofilm cells were treated with imipenem and colistin. Downregulation of these genes affects protein synthesis, which in turn slows down cell metabolism and makes biofilm cells more tolerant to antibiotics. In this investigation, we also found that 5 of 138 small RNAs (sRNAs) were differentially expressed in biofilm regardless of antibiotic treatment or not. Of these, sRNA00203 showed the highest expression levels in biofilm. sRNAs regulate gene expression and are associated with biofilm formation, which may in turn affect the expression of biofilm-specific antibiotic resistance. In summary, when biofilm cells were exposed to sub-MIC doses of colistin and imipenem, coordinated gene responses result in increased biofilm production, multidrug efflux pump expression, and the slowdown of metabolism, which leads to drug tolerance in biofilm. Targeting antibiotic-induced or repressed biofilm-specific genes represents a new strategy for the development of innovative and effective treatments for biofilm-associated infections caused by A. baumannii.

The Anti-Virulence Effect of Sub-Minimal Inhibitory Concentrations of Levofloxacin on Hypervirulent Klebsiella pneumoniae.

BackgroundHypervirulent Klebsiella pneumoniae (hvKp) is best described as a virulent pathogen and generally associated with the hypermucoviscosity phenotype. Increased capsule and aerobactin production are established important hvKp-specific virulence factors. Although hvKp strains have been relatively susceptible to antimicrobials, given the high morbidity and mortality, there is a critical need for alternative strategies for the treatment of hvKp infections. Thus, the anti-virulence therapy has been targeted for the hvKp development of therapeutics.Materials and MethodsFour hvKp isolates with hypermucoviscous phenotype were used in our experiments. Mucoviscosity of the capsule can be assessed by low-speed centrifugation of cultures. CPS amount was determined by glucuronic acid content. The capsule thickness was measured under microscope after ink staining. The transcriptions of gene were measured by quantitative real-time PCR (qRT-PCR). The effect of levofloxacin on the resistance of K. pneumoniae to phagocytosis by macrophages and mouse lethality assay was observed.ResultsOur data revealed that sub-Minimal Inhibitory Concentrations (sub-MIC) of LVX reduce mucoviscosity and CPS production of hvKp. Microscopic observations demonstrated that the capsule of hvkp bacteria became thinned after treatment with LVX. qRT-PCR showed decreased transcript levels of rmpA, wzi, magA, iroN and icuA genes. Down-regulation of these virulence genes occurred leading to increased susceptibility to phagocytosis by macrophages. Mouse lethality assay revealed that the wild strain had the LD50 of 103 CFU, while the sub-MIC LVX-treated bacteria had the LD50 of 105 CFU.ConclusionOur data suggested that LVX may serve as a potential anti-virulence agent for refractory infection by hvKp.

Inhibition of Quorum Sensing Regulated Virulence Factors and Biofilm Formation by Eucalyptus globulus against Multidrug-Resistant Pseudomonas aeruginosa.

ObjectivesThe quorum-sensing–inhibitory and anti-biofilm activities of the methanol extract of E. globulus leaves were determined against clinically isolated multidrug-resistant Pseudomonas aeruginosa.MethodsThe preliminary anti-quorum–sensing (AQS) activity of eucalyptus was investigated against a biosensor strain Chromobacterium violaceum ATCC 12472 (CV12472) by using the agar well diffusion method. The effect of sub-minimum inhibitory concentrations (sub-MICs) of the methanol extract of eucalyptus on different quorum-sensing–regulated virulence factors, such as swarming motility, pyocyanin pigment, exopolysaccharide (EPS), and biofilm formation, against clinical isolates (CIs 2, 3, and 4) and reference PA01 of Pseudomonas aeruginosa were determined using the swarm diameter (mm)-measurement method, chloroform extraction method, phenol (5%)-sulphuric acid (concentrated) method, and the microtiter plate assay respectively, and the inhibition (%) in formation were calculated.ResultsThe preliminary AQS activity (violacein pigment inhibition) of eucalyptus was confirmed against Chromobacterium violaceum ATCC 12472 (CV12472). The eucalyptus extract also showed concentration-dependent inhibition (%) of swarming motility, pyocyanin pigment, EPS, and biofilm formation in different CIs and PA01 of P. aeruginosa.ConclusionOur results revealed the effectiveness of the E. globulus extract for the regulation of quorum-sensing–dependent virulence factors and biofilm formation at a reduced dose (sub-MICs) and suggest that E. globulus may be a therapeutic agent for curing and controlling bacterial infection and thereby reducing the possibility of resistance development in pathogenic strains.

Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa.

Background and Objectives:Antibiotics at sub-minimum inhibitory concentrations (sub-MIC) may alter bacterial virulence factors. The objective of this study was to investigate the effect of gentamicin at sub-MIC concentrations on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa.Materials and Methods:The broth microdilution method was used to determine the MIC of gentamicin for three P. aeruginosa clinical isolates (P1-P3) and standard strains (PAO1 and 8821M). Alginate production and biofilm formation of the bacteria in the presence and absence of sub-MIC concentrations of gentamicin were measured using microtiter plate and carbazole assay, respectively. The real-time PCR method was used to determine the effect of gentamicin at sub-MIC concentrations on the expression level of genes involved in biofilm formation (pelA and pslA) and alginate production (algD and algU).Results:Gentamicin at sub-MIC concentrations significantly reduced alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes in clinical isolate P1. This inhibitory effect was also observed on the alginate production of 8821M strain and biofilm formation of PAO1strain. In clinical isolates, P2 and P3, alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes were significantly increased in exposure to sub-MIC concentrations of gentamicin.Conclusion:This study showed that different phenotypic changes in clinical isolates and standard strains of P. aeruginosa in exposure to sub-MIC concentrations of gentamicin are associated with changes in the expression of virulence genes. Further researches are required to understand the mechanisms involved in regulating the expression of virulence genes after exposure to sub-MIC concentrations of antibiotics.

Antibacterial effect of Satureja hortensis and Salvia officinalis essential oils against major bovine mastitis bacteria

Treatment of bacterial diseases such as bovine mastitis with antibiotics has problems such as antibiotic resistance and drug residue in animal products. Essential oil of medicinal plants have antibacterial activity and are suitable alternatives. This study examined the antimicrobial activity of Salvia officinalis (sage) and Satureja hortensis (savory) essential oils on major mastitis-causing bacteria, including Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. Chemical compositions of essential oils were determined by gas chromatography-mass spectrometry. Minimum inhibitory concentration and minimum bactericidal concentration of oils were determined with serial broth dilution method using autoclaved whole milk rather than synthetic broth. The effect of sub-minimum inhibitory concentrations of essential oils on the growth curve of tested bacteria in milk was obtained in 0, 1, 2, 4, 10, and 24 hours. Major compositions of sage and savory essential oils were carvacrol (61.01%), thymol (20.41%), 1R-α-pinene (7.88%), eucalyptol (32.45%), thymol (28.24%), and α-pinene (13.42%), respectively. The minimum inhibitory concentration and minimum bactericidal concentration ranged 1.25-2.5% and 2.5-5% for savory, and 0.625-1.25% and 1.25-2.5% for sage, respectively. Savory and sage significantly decreased the S. aureus and S. agalactiae population in 4, 10, and 24 h (p < 0.05) and E. coli population in 10 and 24 h (p = 0.01). The sage and savory essential oils had antibacterial effects against three tested bacteria, and sage had a stronger effect than savory because of stronger antibacterial components (carvacrol and thymol). Further in vivo tests are recommended to evaluate the efficiency of these essential oils on the treatment of bovine mastitis.

In vitro effects of sub‐inhibitory concentrations of amoxicillin on physiological responses and virulence determinants in a commensal strain of Escherichia coli

The goal was to study the effects of sub-minimum inhibitory concentrations (sub-MICs) of amoxicillin (AMX) on various physiological responses and virulence determinants in a commensal strain of Escherichia coli. The commensal strain was passaged under various sub-MICs of AMX and its effect on bacterial growth, motility, biofilm formation, expression of outer membrane proteins (OMPs) and cell adhesion was analysed. Bacterial growth was diminished at 1/2 and 1/4 MICs of AMX with significant reduction in growth rate. Using crystal violet (CV) assays and quantification of surface polysaccharides we observed strong biofilm formation, together with reduced swimming motility in E. coli at 1/2 MIC of AMX. Differential OMP expression upon AMX sub-MIC exposure coincided with enhanced cell adhesion to HT-29 cells in vitro. The results demonstrated that sub-MICs of AMX can stimulate unpredictable changes in commensal bacterial strains which can be a potent source for the propagation of antibiotic resistance. The study reports that AMX at 1/2 MIC significantly compromised bacterial growth and swimming motility, alongside inducing biofilm formation. This was also accompanied by upregulation of a single OMP which subsequently increased cell adhesion capabilities in E. coli at 1/2 MIC, thereby enhancing its colonization and survival abilities within the gut microsphere. For the first time, the effects of AMX sub-MICs on a commensal E. coli strain were described. The results corroborate on how antibiotics can act as stimulatory molecules and determine the pathogenicity of commensal bacteria in vivo that can disseminate resistance to other intestinal pathogens or microbes.

Investigation of Pseudomonas aeruginosa Biofilm Formation and Quorum Sensing Genes in Piperacillin/Tazobactam and Ciprofloxacin Sub-minimal Inhibitory Concentrations

Pseudomonas aeruginosa is a non-fermentative, oxidase-positive, motile gram-negative bacillus widespread in nature. The virulence factors of P.aeruginosa including the ability to grow under minimal growth conditions, the widespread presence in nature, and the ability to form biofilms make P.aeruginosa a highly important bacterium along with its resistance mechanisms against many antibiotics. The ability to form biofilms increases the symptom severity in diseases caused by P.aeruginosa and causes difficulties in the treatment. The aim of this study was to investigate the effects of sub-minimal inhibitory concentrations (sub-MIC) of piperacillin/tazobactam (TZP) and ciprofloxacin (CIP) which are used for the treatment of P.aeruginosa infections on biofilm formation and to investigate the relationship between the severity of biofilm formation and Quorum Sensing (QS) genes. The study included 24 P.aeruginosa isolates from the culture collection of Medical Microbiology Laboratory of Gazi University Faculty of Medicine. MIC values of TZP and CIP antibiotics were determined by the microdilution method. The biofilm layers in the antibiotic-free medium and in the sub-MIC (MIC/2, MIC/4 ve MIC/8) concentrations of antibiotics were visualized by using a scanning electron microscope (SEM). The QS genes (lasI, lasR, rhlI, and rhlR) of the 24 isolates with known biofilm characteristics were identified via the amplification of chromosomal DNA by using PCR method. In the study, it was foundthat both antibiotics reduced biofilm formation in a dose-dependent manner in sub-MIC concentrations compared to the antibiotic-free condition and that MIC/2 was the concentration, which reduced the biofilm formation most. These results were further confirmed by viewing the SEM images. The QS genes lasI, lasR, and rhlI were detected in a total of 19 isolates with moderately strong and strong biofilm formation, the rhlR gene was detected in six of the strong biofilm-forming isolates, in four of the moderately strong biofilm-forming isolates, and in three of the weak biofilm-forming isolates, respectively. The investigation of the effects of sub-MIC concentrations of antimicrobials, used for the treatment of P.aeruginosa infections, on the biofilm formation of P.aeruginosa and the investigation and better understanding of the QS systems associated with biofilm production will allow for finding out new treatment approaches and offer different options in combating infections with high morbidity and mortality.

Effect of Sub-Minimum Inhibitory Concentrations of Tyrosol and EDTA on Quorum Sensing and Virulence of Pseudomonas aeruginosa.

IntroductionPseudomonas aeruginosa is considered a dangerous pathogen, as it causes many human diseases, besides that it is resistant to almost all types of antibacterial agents. So, new strategies to overcome P. aeruginosa infection have evolved to attenuate its virulence factors and inhibit its quorum-sensing (QS) activity.PurposeThis study investigated the effect of tyrosol and EDTA as anti-quorum-sensing and antivirulence agents against P. aeruginosa PAO1.MethodsAnti-quorum activity of sub-minimum inhibitory concentrations (sub-MICs) of tyrosol and EDTA was tested using Chromobacterium violaceum (CV 12,472) biosensor bioassay. Miller assay was used to assess the inhibition of QS signal molecules by β-galactosidase activity determination. Also, their effects on the production of protease, lipase, lecithinase, and motility were tested. The inhibitory effects of these molecules on QS regulatory genes and exotoxins genes expression were evaluated by real-time PCR.ResultsTyrosol and EDTA at sub-MICs inhibited the production of violacein pigment. Both compounds inhibited QS molecules production and their associated virulence factors (protease, lipase, lecithinase, and motility) (P≤ 0.05). Besides, the expression levels of QS regulatory genes (lasI, lasR, rhƖI, rhIR, pqsA, and pqsR) and exotoxins genes (exoS and exoY) were significantly reduced (P≤ 0.05).ConclusionBoth tyrosol and EDTA can be used to fight P. aeruginosa infection as anti-quorum-sensing and antivirulence agents at their sub-MICs.

The effect of the sub-minimal inhibitory concentration and the concentrations within resistant mutation window of ciprofloxacin on MIC, swimming motility and biofilm formation of Pseudomonas aeruginosa

ObjectiveTo explore the effect of sub-minimal inhibitory concentration (sub-MIC) and concentrations within resistant mutation window (MSW) of ciprofloxacin (CIP) on minimal inhibitory concentration (MIC), swimming motility and biofilm formation of Pseudomonas aeruginosa, and also to investigate the correlation between swimming motility and genes expression of lasI, lasR, rhlI, rhlR and pqsR. MethodsThe collected strains were incubated under four different concentrations for 5 days. The MIC and mutant prevention concentration (MPC) were measured by the agar dilution method. The diameter of turbid cycle was used to signify the swimming motility. The biofilm formation was measured by the crystal violet stain method. The genes expression of lasI, lasR, rhlI, rhlR and pqsR were measured by RT-PCR. ResultsA total of 11 P. aeruginosa which sensitive to CIP were collected. The incubation within concentrations of MSW made MICs to CIP increased more obviously than under sub-MIC (P < 0.05). The swimming motility showed a trend of being inhibited first and then promoted over time under sub-MIC (P < 0.05), whereas, it was promoted under concentrations within MSW. The biofilm formation was significantly promoted under the concentration of 4×MIC (P < 0.05). Under sub-MIC, the genes expression of rhlR and pqsR had a middle level positive correlation with the promotion of the swimming motility (P < 0.05, r = 0.788 and P < 0.05, r = 0.652, respectively). ConclusionsUnder the concentration of sub-MIC (0.5×MIC) and the concentrations within MSW (1×MIC, 2×MIC and 4×MIC), the effect of CIP on MICs, swimming motility and biofilm formation of P.aeruginosa was quite different. The genes expression of rhlR and pqsR had a middle level positive correlation with the promotion of the swimming motility.

The effect of subminimal inhibitory concentrations of antibiotics on the adherence ability of Pseudomonas aeruginosa to epithelial cells in vitro

Background and purpose: The aim of this study was to examine the influence of subminimal inhibitory concentrations (subMICs) of ceftazidime, ciprofloxacin and gentamicin on the adherence ability and morphology of wild-type Pseudomonas aeruginosa strains to the Buffalo green monkey kidney cell line, using indirect immunofluorescence staining. Materials and methods: Bacterial adherence changes were tested before and after exposure to 1/2, 1/4, 1/8, 1/16 and 1/32 MIC of antibiotics. Results: A statistical difference in the number of attached bacteria after exposure to all subMICs of ceftazidime and ciprofloxacin was observed (p<0.05), even after only 1/2 MIC of gentamicin. Conclusion: The results of this study have shown that antibiotics in much lower concentrations than those necessary for inhibition of bacterial multiplications could damage the adherence of Pseudomonas aeruginosa to the epithelial cell line.

Inhibitory effects of azithromycin on the adherence ability of Porphyromonas gingivalis

Porphyromonas gingivalis is a major pathogen and has a high detection rate in periodontal disease. Fimbriae and hemagglutinin are expressed by P. gingivalis, and these play an important role in the adherence of the bacteria to periodontal tissue and biofilm formation. The aim of this study was to investigate the effects of sub-minimal inhibitory concentrations (sub-MICs) of azithromycin on the adherence of P. gingivalis, focusing on the inhibition of fimbriae expression and hemagglutinin activity. P. gingivalis ATCC 33277 were incubated anaerobically with sub-MICs of azithromycin at 37°C by gentle shaking for 18 hours. The bacterial cells were harvested, washed twice with phosphate-buffered saline (PBS), and the proteins analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Adherence assay and hemagglutinin activity tests were done with the same culture. The results of SDS-PAGE indicated that the sub-MICs of azithromycin inhibited 41-kDa fimbrial protein expression and hemagglutinin activities. The disappearance of 41-kDa fimbrial protein expression and long fimbriae in 0.4µg/mL, 0.2µg/mL, and 0.1µg/mL of azithromycin was confirmed by western blotting and transmission electron microscopy. The adherence of P. gingivalis to human gingival epithelial cells was reduced by sub-MICs of azithromycin compared with the adherence levels without antibiotic. These results suggest that sub-MICs of azithromycin may reduce the adherence of P. gingivalis to host cells, by inhibiting production of fimbriae and hemagglutinin activities. Therefore, azithromycin can be used as a biofilm treatment of periodontal disease caused by P. gingivalis.

Antibiofilm effect of green engineered silver nanoparticles fabricated from Artemisia scoporia extract on the expression of icaA and icaR genes against multidrug-resistant Staphylococcus aureus.

Silver nanoparticles (AgNPs) are at the forefront of the swiftly developing scope of nanotechnology. In the current study, we investigated the green synthesis of AgNPs using Artemisia scoporia as a reducing and capping agent. The biosynthesized AgNPs were characterized using ultraviolet-visible spectroscopy, X-ray diffraction, Fourier-Transform infrared spectroscopy, dispersive absorption spectroscopy, scanning electron microscopy, and transmission electron microscopy. The efficacy of the nanoparticle synthesis was assessed by comparing the antibiofilm activity with commercial AgNPs. The effect of sub-minimum inhibitory concentrations (MICs) of AgNPs on biofilm formation was determined by microtiter plate assay. The expression level of the icaA and icaR genes was assessed by real-time polymerase chain reaction assay. The structural and functional aspects of AgNPs were confirmed. The expression levels of icaA and icaR in the isolates exposed to sub-MIC of both commercial and biosynthetic AgNPs were lower and higher than in the control group, respectively. Our results also indicated that greater reduction and induction in icaA and icaR gene expression were noticed with the sub-MIC doses of biosynthetic AgNP versus commercial AgNP, respectively. This study suggested the application of AgNPs as a significant therapeutic and clinical option in the future and usage for fabricating medical implants. Nevertheless, further investigation is required for examining the pharmaceutical and medicinal properties of AgNPs.

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli

ObjectiveTo evaluate the influence of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on biofilm formation and virulence factors of Escherichia coli clinical isolates. MethodsSub-MICs of CIP were determined using growth curve experiments. The biofilm-forming capacity of E. coli clinical isolates and E. coli ATCC 25922 treated or untreated with sub-MICs of CIP was assessed using a crystal violet staining assay. The biofilm structure of E. coli isolate was assessed with scanning electron microscopy (SEM). The expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were measured using quantification RT-PCR (qRT-PCR) in E. coli isolates and E. coli ATCC 25922. ResultsBased on our results, the sub-MICs of CIP were 1/4 MICs. Sub-MICs of CIP significantly inhibited biofilm formation of E. coli clinical isolates and E. coli ATCC 25922 (p<0.01). SEM analyses indicated that the biofilm structure of the E. coli changed significantly after treatment with sub-MICs of CIP. Expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were also suppressed. ConclusionsThe results revealed that treatment with sub-MICs of CIP for 24h inhibited biofilm formation and reduced the expression of virulence genes and biofilm formation genes in E. coli.

Effects of sub-minimal inhibitory concentrations of antibiotics on the morphology and surface hydrophobicity of periodontopathic anaerobes

It has been reported that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are capable of altering bacterial surface properties and phenotype. In this study, the effects of sub-MICs of certain antibiotics on surface hydrophobicity, cell morphology, and protein profile were ascertained using Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola strains, which are pathogenic bacterial species in periodontal diseases. The MICs of antibiotics were determined by culturing bacteria in media supplemented with serially diluted antibiotic solutions, and sub-MIC of antibiotics was used. The effect of sub-MIC of antibiotics on cell morphology was determined by scanning electron microscopy. Microscopic observation of F. nucleatum and P. gingivalis grown at a sub-MIC of amoxicillin revealed cell enlargement. T. denticola grown at a sub-MIC of doxycycline also showed cell elongation. The relative surface hydrophobicity determined by measuring the ability of the bacteria to absorb n-hexadecane revealed an increase in surface hydrophobicity of F. nucleatum grown at sub-MIC of penicillin and amoxicillin, but a decrease with metronidazole; whereas increased hydrophobicity was observed in T. denticola grown at sub-MIC of doxycycline, metronidazole and tetracycline. The surface hydrophobicity of P. gingivalis increased only when grown in sub-MIC of metronidazole. The protein expression profile of the treated bacteria differed from their respective controls. These results confirmed that sub-MIC concentrations of antibiotics can affect the phenotype, surface properties and morphology of periodontal pathogenic anaerobic bacteria.

Effects of Sub-Minimum Inhibitory Concentrations of Silver Nanoparticles on Some Virulence Factors of Staphylococcus aureus

Effects of Sub-Minimum Inhibitory Concentrations of Silver Nanoparticles on Some Virulence Factors of Staphylococcus aureus

Effects of sub-minimum inhibitory concentrations of lemon essential oil on the acid tolerance and biofilm formation of Streptococcus mutans

ObjectivesLemon essential oil (LEO) is a kind of secondary metabolite from lemon peels and has been found to inhibit cariogenic bacteria for decades. However, its effects on main cariogenic virulence factors are rarely reported. The present study aimed to investigate the effects of sub-minimum inhibitory concentrations (sub-MICs) of LEO on the acid tolerance and biofilm formation of Streptococcus mutans (S. mutans) and preliminarily reveal the possible underlying mechanisms. DesignsEffects of LEO on the acid tolerance and biofilm formation of S. mutans were investigated by the broth dilution method and crystal violet staining method respectively. The expression of luxS, srtA and spaP gene was also determined to explore the underlying mechanism. In addition, Tea polyphenols (TP), a major natural inhibitor of cariogenic virulence factors, and limonene (LIM), the major component of LEO, were selected as comparisons to evaluate the effects of LEO. ResultsSub-MICs of LEO, LIM and TP exhibited a dose-dependent inhibition of growth of S. mutans at pH ranging from 4.0 to 7.0. The formation of S. mutans biofilm was remarkably inhibited and the inhibitory rates of LEO, LIM and TP were 97.87%, 94.88% and 96.01% respectively at 1/2 MIC. Similarly, a down-regulation was observed in the expression of luxS, srtA and spaP gene at sub-MIC levels. ConclusionsEffects of LEO were similar or slightly stronger than LIM and TP, suggesting that LEO might represent a novel, natural anticarious agent that inhibited the specific genes associated with bacterial acid tolerance and biofilm formation without necessarily affecting the growth of oral bacteria.

In vitro effect of subminimal inhibitory concentrations of antibiotics on the biofilm formation ability of Acinetobacter baumannii clinical isolates

The ability of A cinetobacter baumannii strains to form biofilm is one of the most important virulence factor which enables bacterial survival in a harsh environment and decreases antibiotic concentration as well. Subminimal inhibitory concentrations (subMICs) of antibiotics may change bacterial ultrastructure or have an influence on some different molecular mechanisms resulting in morphological or physiological changes in bacteria itself. The aim of this study was to determine effects of 1/2, 1/4, 1/8 and 1/16 minimal inhibitory concentrationsof imipenem, ampicillin-sulbactam, azithromycin, rifampicin and colistin on biofilm formation ability of 22 biofilm non-producing and 46 biofilm producing A. baumannii strains (30 weak producing strains and 16 moderate producing strains). Results of this study indicate that 1/2–1/16 MICs of imipenem, azithromycin, and rifampicin can reduce bacterial biofilm formation ability in moderate producing strains (p < 0.05), whereas 1/16 MIC of imipenem and 1/4–1/8 MICs of rifampicin reduce the biofilm formation in weak producing strains (p < 0.05). Statisticaly significant effect was detected among biofilm non-producing strains after their exposure to 1/16 MIC of azithromycin (p = 0.039). SubMICs of ampicillin-sulbactam and colistin did not have any significant effect on biofilm formation among tested A. baumannii strains.