Strong Biofilm-forming Strains Research Articles

The Staphylococcus aureus ArlS Kinase Inhibitor Tilmicosin Has Potent Anti-Biofilm Activity in Both Static and Flow Conditions.

Staphylococcus aureus can form biofilms on biotic surfaces or implanted materials, leading to biofilm-associated diseases in humans and animals that are refractory to conventional antibiotic treatment. Recent studies indicate that the unique ArlRS regulatory system in S. aureus is a promising target for screening inhibitors that may eradicate formed biofilms, retard virulence and break antimicrobial resistance. In this study, by screening in the library of FDA-approved drugs, tilmicosin was found to inhibit ArlS histidine kinase activity (IC50 = 1.09 μM). By constructing a promoter-fluorescence reporter system, we found that tilmicosin at a concentration of 0.75 μM or 1.5 μM displayed strong inhibition on the expression of the ArlRS regulon genes spx and mgrA in the S. aureus USA300 strain. Microplate assay and confocal laser scanning microscopy showed that tilmicosin at a sub-minimal inhibitory concentration (MIC) had a potent inhibitory effect on biofilms formed by multiple S. aureus strains and a strong biofilm-forming strain of S. epidermidis. In addition, tilmicosin at three-fold of MIC disrupted USA300 mature biofilms and had a strong bactericidal effect on embedded bacteria. Furthermore, in a BioFlux flow biofilm assay, tilmicosin showed potent anti-biofilm activity and synergized with oxacillin against USA300.

Effects of pH, sodium chloride, and temperature on the growth of Listeria monocytogenes biofilms

AbstractListeria monocytogenes is able to form biofilms on food contact surfaces. Effectiveness of salt concentration, pH, and temperature on the formation of L. monocytogenes biofilms was evaluated individually and in combinations using microtiter plate assay by measuring the optical density. The tested strains differed in their biofilm formation (low, moderate, and strong) ability. At 37 °C, decreasing amounts of biofilms was observed in almost all L. monocytogenes strains when the NaCl concentration increased from 0.05 to 15%, but all strains were able to form biofilm even at 1 °C. There was no significant difference in biofilm formation between pH 4, 5, and 6, except for some strains. When stress conditions were tested in combination, the addition of 15% NaCl significantly inhibited the growth of L. monocytogenes at 1 °C and 4 °C, and the weak biofilm-forming strains were less sensitive to the temperature and to NaCl treatments than the strong biofilm-forming strains. These results enhance our knowledge of the application of NaCl, temperature, and pH stresses in the food industry and provide basis to develop new strategies for control of biofilm formation of this pathogen.

Genome Analysis of Pseudomonas aeruginosa Strains from Chronically Infected Patients with High Levels of Persister Formation

The appearance of persister cells with low metabolic rates are key factors leading to antibiotic treatment failure. Such persisters are multidrug tolerant and play a key role in the recalcitrance of biofilm-based chronic infections. Here, we present the genomic analyses of three distinct Pseudomonas aeruginosa Egyptian persister-isolates recovered from chronic human infections. To calculate the persister frequencies, viable counts were determined before and after treatment with levofloxacin. The susceptibilities of isolates to different antibiotics were determined using the agar-dilution method. To determine their recalcitrance, the levofloxacin persisters were further challenged with lethal concentrations of meropenem, tobramycin, or colistin. Furthermore, the biofilm formation of the persister strains was estimated phenotypically, and they were reported to be strong biofilm-forming strains. The genotypic characterization of the persisters was performed using whole genome sequencing (WGS) followed by phylogenetic analysis and resistome profiling. Interestingly, out of the thirty-eight clinical isolates, three isolates (8%) demonstrated a persister phenotype. The three levofloxacin-persister isolates were tested for their susceptibility to selected antibiotics; all of the tested isolates were multidrug resistant (MDR). Additionally, the P. aeruginosa persisters were capable of surviving over 24 h and were not eradicated after exposure to 100X-MIC of levofloxacin. WGS for the three persisters revealed a smaller genome size compared to PAO1-genome. Resistome profiling indicated the presence of a broad collection of antibiotic-resistance genes, including genes encoding for antibiotic-modifying enzymes and efflux pump. Phylogenetic analysis indicated that the persister isolates belong to a distinct clade rather than the deposited P. aeruginosa strains in the GenBank. Conclusively, the persister isolates in our study are MDR and form a highly strong biofilm. WGS revealed a smaller genome that belongs to a distinct clade.

The Correlation Between Biofilm-Forming Ability of Community-Acquired Methicillin-Resistant Staphylococcus aureus Isolated from the Respiratory Tract and Clinical Characteristics in Children.

ObjectiveThis study aimed to investigate the biofilm-forming ability, molecular typing, and antimicrobial resistance of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains isolated from the respiratory tract of children and their correlation with clinical characteristics.MethodsAll CA-MRSA strains were isolated from hospitalized children, and their presentation, molecular typing, antimicrobial susceptibility, and biofilm formation were investigated. The clinical characteristics were compared between the strong and weak biofilm producer groups.ResultsFifty-three CA-MRSA strains were isolated from the respiratory samples of 53 children, with nearly half of them being young infants (0–12 months). Approximately, 88.7% (47/53) of the isolates were resistant to four or more antibiotics, mainly β-lactam antibiotics, lincosamides, and macrolides. Twelve sequence types (STs) and 20 subtypes of staphylococcal protein A (spa) typing were identified, with ST59-t437 (39.6%, 21/53) as the predominant subtype. All strains showed the ability to form biofilms. When compared to children with weak biofilm-forming CA-MRSA strains, those with strong biofilm-forming strains had higher proportions of lower respiratory tract infections (LRTI) (88.5% vs 59.3%), obvious cough symptoms (84.6% vs 51.9%), and severe chest imaging manifestations (76.9% vs 37.0%). Furthermore, a strong biofilm-forming ability significantly increased the risk of prolonged cough in children with LRTI (44.4% vs 14.3%), and a positive correlation between the duration of cough and the extent of biofilm formation was observed. Medical history investigation revealed that the strong biofilm-forming group had a much higher percentage of macrolides intake than the weak biofilm-forming group in the last month before admission (61.5% vs 14.8%).ConclusionST59-t437 was the most prevalent clone in CA-MRSA respiratory isolates among the hospitalized children. All CA-MRSA strains formed biofilms. The stronger the biofilm-forming ability, the more serious and prolonged were the respiratory symptoms.

Surface proteins and the formation of biofilms by Staphylococcus aureus

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12–24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [15N]glycine and l-[1-13C]threonine, or in separate experiments, by (ii) l-[2-13C,15N]leucine. We then measured 13C-15N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15N isotopic enrichments and from the routing of 13C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion.

Hyicin 4244, the first sactibiotic described in staphylococci, exhibits an anti-staphylococcal biofilm activity

Hyicin 4244 is a small antimicrobial peptide with a broad spectrum of activity that was found in the culture supernatant of Staphylococcus hyicus 4244, the genome of which was then sequenced. The bacteriocin gene cluster (hyiSABCDEFG) was mined from its single chromosome and exhibited a genetic organization similar to that of subtilosin A. All genes involved in hyicin 4244 biosynthesis proved to be transcribed and encode proteins that share at least 42% similarity to proteins encoded by the subtilosin A gene cluster. Due to its resemblance to subtilosin A and the presence of three thioether bonds in its structure, hyicin 4244 is assumed to be a 35–amino acid circular sactibiotic, the first to be described in staphylococci. Hyicin 4244 inhibited 14 staphylococcal isolates from either human infections or bovine mastitis, all biofilm formers. Hyicin 4244 significantly reduced the number of colony–forming units (CFU) and the biofilm formation by two strong biofilm-forming strains randomly chosen as representatives of the strains involved in human infections and bovine mastitis. It also reduced the proliferation and viability of sessile cells in established biofilms. Therefore, hyicin 4244 proved not only to prevent biofilm formation by planktonic cells, but also to penetrate the biofilm matrix in vitro, exerting bactericidal activity against staphylococcal sessile cells. This bacteriocin has the potential to become an alternative antimicrobial for either prevention or treatment of biofilm-related infections caused by different staphylococcal species.

ANTIBIOFILM EFFECTS of Citrus limonum and Zingiber officinale Oils on BIOFILM FORMATION of Klebsiella ornithinolytica, Klebsiella oxytoca and Klebsiella terrigena SPECIES.

Background:Microbial cells growing in biofilms, play a huge role in the spread of antimicrobial resistance. In this study, biofilm formation of Klebsiella strains belonging to 3 different Klebsiella species (K. ornithinolytica, K. oxytoca and K. terrigena), cooccurences’ effect on biofilm formation amount and anti-biofilm effects of Citrus limon and Zingiber officinale essential oils on biofilm formations of highest biofilm forming K. ornithinolytica, K. oxytoca and K. terrigena strains were determined.Materials and Methods:Anti-biofilm effects of Citrus limon and Zingiber officinale essential oils on biofilm formations of highest biofilm forming K. ornithinolytica, K. oxytoca and K. terrigena strains were investigated.Results:57% of K. ornithinolytica strains and 50% of K. oxytoca strains were found as Strong Biofilm Forming (SBF), there wasn’t any SBF strain in K. terrigena species. In addition to this, clinical materials of urine and sperm were found as the most frequent clinical materials for strong biofilm forming K. ornithinolytica and K. oxytoca isolations respectively (63%; 100%) Secondly, all K. ornithinolytica strains isolated from surgical intensive care unit and all K. oxytoca strains isolated from service units of urology were found as SBF. Apart from these, although the amount of biofilm, formed by co-occurence of K. ornithinolytica - K. oxytoca and K. oxytoca - K. terrigena were more than the amount ofbiofilm formed by themselves separately, biofilm formation amount of co-occurrence of K. ornitholytica - K. terrigena strains was lower than biofilm formation amount of K. ornithinolytica but higher than biofilm formation amount of K. terrigena.Conclusion:The antibiofilm effects of Citrus limonum and Zingiber officinale essential oils could be used against biofilm Klebsiella aquired infections.

Antibacterial and anti-biofilm activities of thiazolidione derivatives against clinical staphylococcus strains

Both Staphylococcus aureus and Staphylococcus epidermidis can form biofilms on natural surfaces or abiotic surfaces, such as medical implants, resulting in biofilm-associated diseases that are refractory to antibiotic treatment. We previously reported a promising antibacterial compound (Compound 2) and its derivatives with bactericidal and anti-biofilm activities against both S. epidermidis and S. aureus. We have further evaluated the antibacterial activities of four Compound 2 derivatives (H2-38, H2-39, H2-74 and H2-81) against 163 clinical strains of S. epidermidis and S. aureus, including methicillin-susceptible and methicillin-resistant strains, as well as biofilm-forming and non-biofilm-forming strains. The four derivatives inhibited the planktonic growth of all of the clinical staphylococcal isolates, including methicillin-resistant S. aureus and methicillin-resistant S. epidermidis and displayed bactericidal activities against both immature (6 h) and mature (24 h) biofilms formed by the strong biofilm-forming strains. The derivatives, which all target YycG, will help us to develop new antimicrobial agents against multidrug-resistant staphylococci infections and biofilm-associated diseases.

Nutrition effects on the biofilm immobilization and 3,5-DNBA degradation of Comamonas testosteroni A3 during bioaugmentation treatment.

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems.

A chalcone with potent inhibiting activity against biofilm formation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, frequently causes biofilm infections. Currently, resistance of bacteria within the biofilm to conventional antimicrobials poses a major obstacle to effective medical treatment on a global scale. Novel agents that are effective against NTHi biofilm are therefore urgently required. In this study, a series of natural and synthetic chalcones with various chemical substituents were evaluated in vitro for their antibiofilm activities against strong biofilm-forming strains of NTHi. Of the test chalcones, 3-hydroxychalcone (chalcone 8) exhibited the most potent inhibitory activity, its mean minimum biofilm inhibitory concentration (MBIC50 ) being 16 μg/mL (71.35 μM), or approximately sixfold more active than the reference drug, azithromycin (MBIC50 419.68 μM). The inhibitory activity of chalcone 8, which is a chemically modified chalcone, appeared to be superior to those of the natural chalcones tested. Significantly, chalcone 8 inhibited biofilm formation by all studied NTHi strains, indicating that the antibiofilm activities of this compound occur across multiple strong-biofilm forming NTHi isolates of different clinical origins. According to antimicrobial and growth curve assays, chalcone 8 at concentrations that decreased biofilm formation did not affect growth of NTHi, suggesting the biofilm inhibitory effect of chalcone 8 is non-antimicrobial. In terms of structure-activity relationship, the possible substituent on the chalcone backbone required for antibiofilm activity is discussed. These findings indicate that 3-hydroxychalcone (chalcone 8) has powerful antibiofilm activity and suggest the potential application of chalcone 8 as a new therapeutic agent for control of NTHi biofilm-associated infections.

Biofilm-forming ability of Staphylococcus aureus strains isolated from human skin

BackgroundStaphylococcus aureus produces various toxins and enzymes, and its presence can exacerbate skin conditions. Previous studies have shown that S. aureus is involved in skin deterioration, even in normal tissue. Biofilm strains show much greater resistance to antimicrobial agents and therefore require a much higher concentration of biocide than planktonic counterparts. ObjectiveAs such, alternative strategies and more effective therapeutic agents against biofilm-producing S. aureus in skin are of great interest. Therefore, we turned our attention to differences in 50 clinical biofilm strains isolated from human facial skin. MethodsBased on S. aureus density on facial skin, we divided donors into two groups: relatively low density (LSG) and high density (HSG). In general, strong biofilm-forming strains were detected in the HSG donors. Two strains from each of the groups were submitted to gene microarray analysis to investigate expression differences and confirmed by RT-PCR. ResultsIn total, 111 of 7775 genes were differentially expressed between low (SA2 and SA7) vs. high (SA10 and SA33) biofilm-forming clinical strains. These genes include already well-known as biofilm formation related genes like icaABCD and lrgAB, and newly identified genes (sdrC, sspBCP) by RT-PCR. Comparison of gene expression differences between the two groups available at NCBI Gene Expression Omnibus accession number GSE44268. ConclusionOur results suggest that S. aureus density in the skin is closely related to biofilm-forming ability, and we have identified several potential target genes that may be involved in regulating biofilm formation in situ.

Susceptibility of clinical isolates of Candida to photodynamic effects of curcumin

The resistance of Candida species to antifungals represents a major challenge for therapeutic and prophylactic strategies. This study evaluated photodynamic therapy (PDT) mediated by Curcumin (CUR) against clinical isolates of C. albicans, C. tropicalis, and C. glabrata, both in planktonic and biofilm forms. Suspensions of Candida were treated with three CUR concentrations and exposed to four LED fluences. The protocol that showed the best outcomes for inactivation of the planktonic phase was selected to be evaluated against Candida biofilms. In addition, two higher CUR concentrations were tested. The metabolic activity of biofilms was evaluated by means of XTT reduction assay and the biofilm biomass was evaluated using crystal violet (CV) staining assay. Data were analyzed in a mixed model nested ANOVA, Wilcoxon's nonparametric tests, and the Kruskal-Wallis test (α = 5%). The use of CUR in association with light was able to promote a significant antifungal effect against the planktonic form of the yeasts. When using 40 µM of CUR, the metabolic activity of C. albicans, C. glabrata, and C. tropicalis biofilms was reduced by 85%, 85%, and 73%, respectively, at 18 J/cm(2) . CUR-mediated PDT also decreased the biofilm biomass of all species evaluated. In addition, CV staining showed that C. albicans isolates were strong biofilm-forming strains, when compared with C. glabrata and C. tropicalis isolates. The results from the present investigation showed that low CUR concentrations can be highly effective for inactivating Candida isolates when associated with light excitation.

Transcriptomic Analysis for Genetic Mechanisms of the Factors Related to Biofilm Formation in Escherichia coli O157:H7

Two lineages of enterohemorrhagic Escherichia coli O157:H7 (EDL933, Stx1(+) and Stx2(+)) and 86-24 (Stx2(+)) were investigated to determine the genetic basis of biofilm formation on abiotic surfaces. Strain EDL933 formed a robust biofilm while strain 86-24 formed almost no biofilm on either polystyrene plates or polyethylene tubes. Whole-transcriptome profiles of EDL933 versus 86-24 revealed that in the strong biofilm-forming strain, genes involved in curli biosynthesis and cellulose production were significantly induced, whereas genes involved in indole signaling were most repressed. Additionally, 49 phage genes were highly induced and repressed between the two strains. Curli assays using Congo red plates and scanning electron microscopy corroborated the microarray data as the EDL933 strain produced a large amount of curli, while strain 86-24 formed much less curli. Moreover, EDL933 produced 19-fold more cellulose than 86-24, and indole production in EDL933 was two times lower than that of the strain 86-24. Therefore, it appears E. coli O157:H7 EDL933 produces more biofilm because of its increased curli and cellulose production and reduced indole production.

Assessment of in vitro biofilm formation by Helicobacter pylori

Biofilms are surface-bound communities of bacterial cells that are implicated in their survival. As with various bacteria studied to date, Helicobacter pylori can have an alternate lifestyle as a biofilm. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. However, the mechanisms of its biofilm development remain unclear. We analyzed the basic characteristics of the biofilm-forming ability in strain TK1402. In order to characterize the biofilm-forming ability of the H. pylori strains, auto-aggregation, motility and hydrophobicity, which are important factors in biofilm formation by other bacteria, were analyzed. Further, we tested whether cell growth participated in biofilm formation in strain TK1402. There were no significant differences in the auto-aggregation, motility and hydrophobicity of strain TK1402 compared with the other strains. On the other hand, pre-culture of this strain for 24-48 h resulted in decreased biofilm formation. TK1402 is a strong biofilm-forming strain of H. pylori in Brucella broth supplemented with 7% fetal calf serum. It is possible that biofilm-forming cell growth is a principal factor in biofilm development.

Biofilm Formation by Multidrug-Resistant Salmonella enterica Serotype Typhimurium Phage Type DT104 and Other Pathogens

The biofilm-forming capability of Salmonella enterica serotypes Typhimurium and Heidelberg, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli O157:H7, Klebsiella pneumoniae, and Acinetobacter baumannii isolated from humans, animal farms, and retail meat products was evaluated by using a microplate assay. The tested bacterial species showed interstrain variation in their capabilities to form biofilms. Strong biofilm-forming strains of S. enterica serotypes, E. coli O157: H7, P. aeruginosa, K. pneumoniae, and A. baumannii were resistant to at least four of the tested antibiotics. To understand their potential in forming biofilms in food-processing environments, the strong biofilm formers grown in beef, turkey, and lettuce broths were further investigated on stainless steel and glass surfaces. Among the tested strains, Salmonella Typhimurium phage type DT104 (Salmonella Typhimurium DT104) isolated from retail beef formed the strongest biofilm on stainless steel and glass in beef and turkey broths. K. pneumoniae, L. monocytogenes, and P. aeruginosa were also able to form strong biofilms on the tested surface materials. Salmonella Typhimurium DT104 developed a biofilm on stainless steel in beef and turkey broths through (i) initial attachment to the surface, (ii) formation of microcolonies, and (iii) biofilm maturation. These findings indicated that Salmonella Typhimurium DT104 along with other bacterial pathogens could be a source of cross-contamination during handling and processing of food.

Transfer of Listeria monocytogenes during Slicing of Turkey Breast, Bologna, and Salami with Simulated Kitchen Knives

In response to continued concerns regarding Listeria cross-contamination during the slicing of deli meats, a series of specially prepared grade 304 and 316 stainless steel kitchen knife blades was inoculated with a six-strain Listeria monocytogenes cocktail (108, 105, and 103 CFU per blade) composed of two weak, two medium, and two strong biofilm-forming strains. The blades were then attached to an Instron 5565 electromechanical compression analyzer and used to slice whole chubs of delicatessen turkey breast, bologna, and salami to entirety (30 slices) at a cutting speed of 8.3 mm/s. Homogenates of the slices in University of Vermont Medium were surface or pour plated with modified Oxford agar and then enriched. Listeria transfer from knife blades inoculated at 108 CFU per blade was logarithmic, with a 2-log decrease seen after 8 to 12 slices and direct counts obtained thereafter out to 30 slices. However, blades containing 105 and 103 CFU per blade typically yielded direct counts out to only 20 and 5 slices, respectively. Normalizing data on a log scale for the first 10 slices resulted in significantly greater Listeria transfer and “tailing” from grade 304 as opposed to grade 316 stainless (P < 0.05) for all three products. After 1 year of use, surface roughness values as determined by surface profilometry were significantly greater (P < 0.001) for grade 304 than for grade 316 stainless blades. Cutting force and blade sharpness were not significantly different (P > 0.05) within stainless steel grade (P < 0.05) for each product. However, significant differences in cutting force were seen between salami and turkey (P < 0.05) for grades 304 and 316 stainless, respectively. In addition to compositional differences in the deli meats and knife blades, wear and scoring on the blade likely affected Listeria transfer during slicing.