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The growth of respiratory diseases, as witnessed through the SARS and COVID-19 outbreaks, and antimicrobial-resistance together pose a serious threat to humanity. One reason for antimicrobial resistance is formation of bacterial biofilms. In this study the sulphated polysaccharides from green algae Chlamydomonas reinhardtii (Cr-SPs) is tested for its antibacterial and antibiofilm potential against Klebsiella pneumoniae and Serratia marcescens. Agar cup assay clearly indicated the antibacterial potential of Cr-SPs. Minimum inhibitory concentration (MIC50) of Cr-SPs against Klebsiella pneumoniae was found to be 850 µg/ml, and it is 800 µg/ml in Serratia marcescens. Time-kill and colony-forming ability assays suggest the concentration-dependent bactericidal potential of Cr-SPs. Cr-SPs showed 74–100% decrease in biofilm formation in a concentration-dependent manner by modifying the cell surface hydrophobic properties of these bacteria. Cr-SPs have also distorted preformed-biofilms by their ability to interact and destroy the extra polymeric substance and eDNA of the matured biofilm. Scanning electron microscopy analysis showed that Cr-SPs effectively altered the morphology of these bacterial cells and distorted the bacterial biofilms. Furthermore reduced protease, urease and prodigiosin pigment production suggest that Cr-SPs interferes the quorum sensing mechanism in these bacteria. The current study paves way towards developing Cr-SPs as a control strategy for treatment of respiratory tract infections.

Pseudomonas putida is widely recognized as a spoiler of fresh foods under cold storage, and recently associated also with infections in clinical settings. The presence of antibiotic resistance genes (ARGs) could be acquired and transmitted by horizontal genetic transfer and further increase the risk associated with its persistence in food and the need to be deeper investigated. Thus, in this work we presented a genomic and phenotypic analysis of the psychrotrophic P. putida ITEM 17297 to provide new insight into AR mechanisms by this species until now widely studied only for its spoilage traits. ITEM 17297 displayed resistance to several classes of antibiotics and it also formed huge amounts of biofilm; this latter registered increases at 15 ​°C in comparison to the optimum growth condition (30 ​°C). After ITEM 17297 biofilms exposure to antibiotic concentrations higher than 10-fold their MIC values no eradication occurred; interestingly, biomasses of biofilm cultivated at 15 ​°C increased their amount in a dose-dependent manner. Genomic analyses revealed determinants (RND-systems, ABC-transporters, and MFS-efflux pumps) for multi-drugs resistance (β-lactams, macrolides, nalidixic acid, tetracycline, fusidic acid and bacitracin) and a novel ampC allele. Biofilm and motility related pathways were depicted underlying their contribution to AR. Based on these results, underestimated psychrotrophic pseudomonas, such as the herein studied ITEM 17297 strain, might assume relevance in relation to the risk associated with the transfer of antimicrobial resistance genes to humans through cold stored contaminated foods. P. putida biofilm and AR related molecular targets herein identified will provide a basis to clarify the interaction between AR and biofilm formation and to develop novel strategies to counteract the persistence of multidrug resistant P. putida in the food chain.

In vitro eradication of the C. albicans and S. mutans mixed biofilms by eugenol alone and in combination with the antimicrobial drugs. Previously characterized strains of C. albicans (CAJ-01 and CAJ-12) and S. mutans MTCC497 were used to evaluate the eradication of biofilms using XTT reduction assay, viability assay, time dependent killing assay and scanning electron microscopy (SEM). Synergistic interaction was assessed by checkerboard method. Sessile MIC (SMIC) of eugenol was equivalent to the planktonic MIC (PMIC) against C. albicans and S. mutans mixed biofilms. SMIC of fluconazole and azithromycin was increased upto 1000-folds over PMIC. Eradication of single or mixed biofilms was evident from the viability assay and SEM. At 1 × MIC of eugenol, log10CFU count of C. albicans cells were decreased from 6.3 to 4.2 and 3.8 (p C. albicans and S. mutans cells from glass surface at 800 µg/mL concentration of eugenol. Time dependent killing assay showed dose dependent effect of eugenol on pre-formed CAJ-01, CAJ-12 and S. mutans biofilm cells. Eugenol was highly synergistic with fluconazole (FICI = 0.156) against CAJ-12 single biofilms. However, the combination of eugenol and azithromycin showed maximum synergy (FICI = 0.140) against pre-formed C. albicans and S. mutans mixed biofilms. These findings highlighted the promising efficacy of eugenol in the eradication of biofilms of two oral pathogens (C. albicans and S. mutans) in vitro and could also be exploited in synergy with fluconazole and azithromycin in controlling oral infections.

The presence of biofilm within a chronic wound may delay the healing process. Thus, control of biofilm formation and providing bactericidal effect are crucial factors for wound healing management. Alginate-based nanocomposite hydrogels have been suggested as dressing materials for wound treatment, which are employed as a biocompatible matrix. Therefore, in this study, we aimed to develop a biocompatible antimicrobial wound dressing containing AgNPs and demonstrate its efficacy against polymicrobial wound biofilms by using a biofilm flow device to simulate a chronic infected, exuding wound and specific wound environment. The results from agar well diffusion, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays showed that TA-AgNPs exhibited antibacterial activity against wound pathogens. Additionally, the Minimum Biofilm Eradication Concentration assay (MBEC) demonstrated it could impair biofilm formation. Importantly, our TA-AgNPs/Alginate hydrogel clearly showed antibacterial activities against Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, we used the biofilm flow device to test the topical antimicrobial hydrogel against a three-species biofilm. We found that TA-AgNPs/Alginate hydrogel significantly showed a 3–4 log reduction in bacterial numbers when applied with multiple doses at 24 h intervals, and was especially effective against the chronic wound pathogen P. aeruginosa. This work highlighted that the TA-AgNPs/Alginate hydrogel is a promising material for treating complex wound biofilms.

Staphylococcus aureus (S. aureus) is considered by the World Health Organization as a high priority pathogen for which new therapies are needed. This is particularly important for biofilm implant-associated infections once the only available treatment option implies a surgical procedure combined with antibiotic therapy. Consequently, these infections represent an economic burden for Healthcare Systems. A new strategy has emerged to tackle this problem: for small bugs, small particles. Here, we describe how nanotechnology-based systems have been studied to treat S. aureus biofilms. Their features, drawbacks and potentialities to impact the treatment of these infections are highlighted. Furthermore, we also outline biofilm models and assays required for preclinical validation of those nanosystems to smooth the process of clinical translation., Staphylococcus aureus biofilms are a major cause for implant-associated infections. The lack of an effective treatment and the escalating numbers of antibiotic-resistance events are prompting the research toward alternative nano-based strategies.

Removal of biofilms is extremely pivotal in environmental and medicinal fields. Therefore, reporting the new-enzymes and their combinations for dispersal of infectious biofilms can be extremely critical. Herein, for the first time, we accessed the enzyme &ldquo, protease from bovine pancreas type-I (PtI)&rdquo, for anti-biofilm properties. We further investigated the anti-biofilm potential of PtI in combination with &alpha, amylase from Bacillus sp. (&alpha, A). PtI showed a very significant biofilm inhibition effect (86.5%, 88.4%, and 67%) and biofilm prevention effect (66%, 64%, and 70%), against the E. coli, S. aureus, and MRSA, respectively. However, the new enzyme combination (Ec-PtI+&alpha, A) exhibited biofilm inhibition effect (78%, 90%, and 93%) and a biofilm prevention effect (44%, 51%, and 77%) against E. coli, S. aureus, and MRSA, respectively. The studied enzymes were found not to be anti-bacterial against the E. coli, S. aureus, and MRSA. In summary, the PtI exhibited significant anti-biofilm effects against S. aureus, MRSA, and E. coli. Ec-PtI+&alpha, A exhibited enhancement of the anti-biofilm effects against S. aureus and MRSA biofilms. Therefore, this study revealed that this Ec-PtI+&alpha, A enzymatic system can be extremely vital for the treatment of biofilm complications resulting from E. coli, S. aureus, and MRSA.

Coagulase-negative staphylococci (CoNS) widely colonize the human skin and play an active role in host defense. However, these bacteria may cause malodours and increase infection incidence rate in immune-compromised patients and individuals with catheters and implants. CoNS spreading is favored by biofilm formation that also promotes the release of virulence factors and drug resistance. Biofilm control or eradication by antimicrobial peptides (AMPs) represents an attractive strategy which is worth investigating. In this work, bovine lactoferrin (BLF) hydrolysate (HLF) was in vitro evaluated for its antimicrobial and antibiofilm activities against skin-related coagulase negative and positive staphylococci. Despite a minimal inhibitory concentration (MIC) recorded for HLF ranging from 10 to more than 20 mg/mL, a minimal biofilm inhibitory concentration (MIBC) equal to 2.5 mg/mL was found for most target strains. Conversely, MIBC values referred to the individual peptides, LFcinB or LFmpin (herein purified and identified) were significantly lower. Finally, the application of 2.5 mg/mL HLF solution by dipping and spraying on biofilm-attached glass surfaces also caused a high biofilm eradication rate depending on the incubation time, thus attracting interest for future applications in cosmetic formulation for skin care.

Pseudomonas aeruginosa biofilms employ a variety of strategies to hijack the host immune defense system to achieve chronic infection. However, the bacterial components that are involved in this process are not yet fully understood. PcrV, a needle tip protein of the P. aeruginosa type III secretion system (T3SS), was downregulated during P. aeruginosa biofilm infection. The impaired expression of the P. aeruginosa pcrV gene is associated with attenuated immune activation and an increased percentage of M2 macrophages following P. aeruginosa biofilm infection. Treatment with exogenous PcrV produced from Escherichia coli elevated tissue inflammation and the percentage of M1 macrophages, resulting in reduction in the biofilm burden. Further analyses demonstrated that the potential of PcrV to induce classically activated M1 macrophages as evidenced by the increased production of proinflammatory cytokines and anti-bacterial mediators, including inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS), as well as increased phagocytosis of bacteria. Mechanistically, PcrV-mediated promotion of macrophage M1 polarization and phagocytosis occurs through the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathways. Collectively, these findings reveal a potential role of PcrV in skewing host immune defense to promote P. aeruginosa biofilm infection and provide new insights into the therapeutic strategies for P. aeruginosa biofilm infection.

Persistent infections, usually associated with biofilm-producing bacteria, are challenging for both medical and scientific communities. The potential interest in drug repurposing for biofilm control is growing due to both disinvestment in antibiotic R&amp, D and reduced efficacy of the available panel of antibiotics. In the present study, the antibacterial and antibiofilm activities of four non-steroidal anti-inflammatory drugs (NSAIDs), piroxicam (PXC), diclofenac sodium (DCF), acetylsalicylic acid (ASA) and naproxen sodium (NPX) were evaluated against Escherichia coli and Staphylococcus aureus. The minimum inhibitory/bactericidal concentrations (MICs and MBCs) and the dose&ndash, response curves from exposure to the selected NSAIDs were determined. MICs were found for PXC (800 &mu, g/mL) and ASA (1750 &mu, g/mL) against E. coli, and for DCF (2000 &mu, g/mL) and ASA (2000 &mu, g/mL) against S. aureus. No MBCs were found (&gt, 2000 &mu, g/mL). The potential of NSAIDs to eradicate preformed biofilms was characterized in terms of biofilm mass, metabolic activity and cell culturability. Additionally, the NSAIDs were tested in combination with kanamycin (KAN) and tetracycline (TET). ASA, DCF and PXC promoted significant reductions in metabolic activity and culturability. However, only PXC promoted biofilm mass removal. Additive interactions were obtained for most of the combinations between NSAIDs and KAN or TET. In general, NSAIDs appear to be a promising strategy to control biofilms as they demonstrated to be more effective than conventional antibiotics.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen which has been listed as a high-priority multi-drug resistance pathogen by the World Health Organization (WHO). Persistent MRSA infections are often associated with biofilm formation and resistance to conventional antimicrobial therapy. Inhibiting the surface adherence and the virulence of the bacterium is the current alternative approach without affecting growth to reduce the possibility of resistance development. Although numerous antibiofilm agents have been identified, their mode of action remains unclear. Combining two drugs with different modes of action will improve the efficiency of the treatment strategy against MRSA. The present study was aimed to decipher the molecular mechanism underlying the antibiofilm activity of thymol against MRSA and assess the ability of thymol to improve the antibacterial activity of rifampicin. Thymol significantly inhibited 88% of MRSA biofilm formation at 100 μg/ml and reduced the surface adherence of MRSA on glass, stainless steel, and titanium surface coated with human plasma as evidenced by microscopic analyses. qPCR analysis of global virulence regulatory genes and biofilm assay with S. aureus wild type, ΔsarA, and Δagr strains revealed the sarA-mediated antibiofilm activity of thymol and inhibition of sarA-controlled virulence factors. Congo red assay and erythrocyte lysis assay further confirmed the reduction in polysaccharide intracellular adhesin and hemolysin. Importantly, thymol enhanced the antibacterial and the biofilm eradication efficiency of rifampicin against MRSA and also reduced the formation of persisters. Thus, the present study reveals the sarA-dependent antibiofilm efficacy of MRSA and suggests thymol as the promising combinatorial candidate in potentiating the antibacterial activity of rifampicin against persistent MRSA infections.

Most biofilms in nature are formed by multiple microbial species, and such mixed-species biofilms represent the actual lifestyles of microbes, including bacteria, fungi, viruses (phages), and/or protozoa. Microorganisms cooperate and compete in mixed-species biofilms. Mixed-species biofilm formation and environmental resistance are major threats to water supply, food industry, and human health. The methods commonly used for microbial eradication, such as antibiotic or disinfectant treatments, are often ineffective for mixed-species biofilm consortia due to their physical matrix barrier and physiological interactions. For the last decade, an increasing number of investigations have been devoted to the usage of cold atmospheric plasma (CAP), which is produced by dielectric barrier discharges or plasma jets to prevent or eliminate microbial biofilms. Here, we summarized the production of CAP, the inactivation of microorganisms upon CAP treatment, and the microbial factors affecting the efficacy of CAP procedure. The applications of CAP as antibiotic alternative strategies for fighting mixed-species biofilms were also addressed.

Staphylococcus aureus is considered one of the leading pathogens responsible for community and healthcare-associated infections. Among them, infections caused by methicillin-resistant strains (MRSA) are connected with ineffective or prolonged treatment. The therapy of staphylococcal infections faces many difficulties, not only because of the bacteria’s resistance to antibiotics and the multiplicity of virulence factors it produces, but also due to its ability to form a biofilm. The present review focuses on several approaches used for the assessment of staphylococcal biofilm eradication. The methods described here are successfully applied in research on the prevention of biofilm-associated infections, as well as in their management. They include not only the evaluation of the antimicrobial activity of novel compounds, but also the methods for biomaterial functionalization. Moreover, the advantages and limitations of different dyes and techniques used for biofilm characterization are discussed. Therefore, this review may be helpful for those scientists who work on the development of new antistaphylococcal compounds.

Hospital-acquired infections are common in burn patients and are the major contributors of morbidity and mortality. Bacterial infections such as Staphylococcus aureus (S. aureus) and Acinetobacter baumannii (A. baumannii) are difficult to treat due to their biofilm formation and rapidly acquiring resistance to antibiotics. This work presents a newly developed hydrogel that has the potential for treating bacterial wound infections. The hydrogel formulation is based on an antimicrobial peptide (AMP), epsilon-poly-l-lysine (EPL) and catechol, which was cross-linked via mussel-inspired chemistry between the amine and phenol groups. In vitro studies showed that EPL-catechol hydrogels possess impressive antimicrobial and antibiofilm properties toward multidrug-resistant A. baumannii (MRAB). In addition, cytotoxicity study with the clonal mouse myoblast cell line (C2C12) revealed the good biocompatibility of this hydrogel. Furthermore, we created a second-degree burn wound on the mice dorsal skin surface followed by contamination with MRAB. Our results showed that the hydrogel significantly reduced the bacterial burden by more than four orders of magnitude in infected burn wounds. Additionally, there was no significant histological alteration with hydrogel application on mice skin. Based on these results, we concluded that EPL-catechol hydrogel is a promising future biomaterial to fight against multidrug-resistant bacterial infections.

Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown., Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here, we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Coadministration of the nitroxide CTEMPO (4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl) with ciprofloxacin significantly improved the biofilm eradication activity of the antibiotic against S. aureus. Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced the ciprofloxacin minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter cells localized at the surface and base of the biofilm structure. No toxicity to human cells was observed for the nitroxide CTEMPO or the nitroxide-antibiotic hybrids. Taken together, our results show that nitroxides can mediate the dispersal of Gram-positive biofilms and that dual-acting biofilm eradication antibiotics may provide broad-spectrum therapies for the treatment of biofilm-related infections.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Contains fulltext : 196638.pdf (Publisher’s version ) (Open Access) Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Bacteriophages are the viruses of bacteria. In the guise of phage therapy they have been used for decades to successfully treat what are probable biofilm-containing chronic bacterial infections. More recently, phage treatment or biocontrol of biofilm bacteria has been brought back to the laboratory for more rigorous assessment as well as towards the use of phages to combat environmental biofilms, ones other than those directly associated with bacterial infections. Considered in a companion article is the inherent ecological utility of bacteriophages versus antibiotics as anti-biofilm agents. Discussed here is a model for phage ecological interaction with bacteria as they may occur across biofilm-containing ecosystems. Specifically, to the extent that individual bacterial types are not highly abundant within biofilm-containing environments, then phage exploitation of those bacteria may represent a “Feast-or-famine” existence in which infection of highly localized concentrations of phage-sensitive bacteria alternate with treacherous searches by the resulting phage progeny virions for new concentrations of phage-sensitive bacteria to infect. An updated synopsis of the literature concerning laboratory testing of phage use to combat bacterial biofilms is then provided along with tips on how “Ecologically” such phage-mediated biofilm control can be modified to more reliably achieve anti-biofilm efficacy.

There is a need for non-antibiotic, antimicrobial compositions with low toxicity capable of broad-spectrum eradication of pathogenic biofilms in food preparation and healthcare settings. In this study we demonstrated complete biofilm eradication within 60 min with synergistic combinations of caprylic and polygalacturonic (PG) acids in an in vitro biofilm eradication model against representative hospital and foodborne infectious pathogen biofilms (methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Salmonella enteritidis). Antimicrobial synergy against biofilms was demonstrated by quantifying viable organisms remaining in biofilms exposed to caprylic acid alone, PG acid alone, or combinations of the two. The combinations also synergistically inhibited growth of planktonic organisms. Toxicity of the combination was assessed in vitro on L929 fibroblasts incubated with extracts of caprylic and PG acid combinations using the Alamar Blue metabolic activity assay and the Trypan Blue exclusion cell viability assay. The extracts did not produce cytotoxic responses relative to untreated control fibroblasts.

Biofilms cause biofouling, pipe plugging, prostheses colonization, disease, and nosocomial infections. Bacterial biofilms are more resilient to sterilization methods than planktonic bacteria; therefore, better control methods are required. The use of gas discharge plasmas is an appropriate alternative because plasmas contain a mixture of reactive agents that are well known for bacterial decontamination. This study assesses culture medium-abiotic surface combinations leading to robust biofilms and tests an air-based coaxial dielectric barrier discharge (DBD) plasma source on Pseudomonas aeruginosa biofilms grown in continuous culture under those selected conditions. Biofilms were eradicated after a 15-min plasma treatment, resulting in a CFU/mL decrease of 5.6 log10 units. CFU/mL decreases of 1.6 and 2.7 log10 units were achieved after a 3-min plasma exposure to ambient and moistened air plasma, respectively, although viability assays showed that some cells were alive. Moistened-air plasma resulted in a faster biofilm inactivation, with decimal reduction times of 1.14 and 4.36 min. The coaxial DBD air-based plasma source presented here is effective for Pseudomonas biofilm inactivation, affordable because it does not rely on expensive gases, and easy to handle for indirect surface treatment. To the best of our knowledge, the search for the best combination medium surface leading to robust biofilms before plasma treatment has not been previously assessed., Centro de Investigación y Desarrollo en Fermentaciones Industriales

The aim was to explore the antimicrobial activity of a synthetic peptide (AMP38) and its synergy with imipenem against imipenem-resistant Pseudomonas aeruginosa. The main mechanism of imipenem resistance is the loss or alteration of protein OprD. Time-kill and minimal biofilm eradication concentration (MBEC) determinations were carried out by using clinical imipenem-resistant strains. AMP38 was markedly synergistic with imipenem when determined in imipenem-resistant P. aeruginosa. MBEC obtained for the combination of AMP38 and imipenem was of 62.5 μg/mL, whereas the MBEC of each antimicrobial separately was 500 μg/mL. AMP38 should be regarded as a promising antimicrobial to fight MDR P. aeruginosa infections. Moreover, killing effect and antibiofilm activity of AMP38 plus imipenem was much higher than that of colistin plus imipenem.