Polymicrobial Interactions

Background and Aim: Candida albicans is the most prevalent human fungal pathogen. In biofilms, C. albicans becomes more resistant to antifungal agents because of the production of an extracellular matrix (ECM) that protects the yeast cells. This study aimed to determine the effects of hydrolase enzymes and the Bgl2 ligand on monomicrobial and polymicrobial biofilms. Materials and Methods: Biofilm induction in rats was carried out using streptomycin (25 mg/kg) and gentamicin (7.5 mg/kg) administered orally once per day for 5 days. Rats were injected subcutaneously with cortisone acetate (225 mg/kg) as an immunosuppressant on day 5. In addition, rats were orally administered C. albicans for the single microbial model and a combination of C. albicans with Escherichia coli for the polymicrobial model. Following the biofilm production, the groups were treated with glucosamine (8.57 mg/kg body weight) and Achatina fulica hydrolases (1.5 mL) orally for 2 weeks. The reduction of the biofilm...

There is a growing interest in natural materials with antibacterial qualities as alternatives. Jatropha curcas latex has long been used to treat infections. Objective: Compare the antimicrobial activities of both Jatropha curcas latex and the used antibiotics against the isolated microorganisms which cause wound burn infection. Methods: An experimental study was carried out in Sana'a City, Yemen, to assess the antimicrobial properties of J. curcas latex. Infected burn wounds were found to contain pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. Standard antimicrobial strains were subjected to testing as well. We obtained latex from verified J. curcas specimens and subjected it to phytochemical analysis. The assessment of antimicrobial activity involved the use of agar well diffusion, disc diffusion, and broth dilution methods to establish inhibition zones and minimum inhibitory concentrations (MICs). Results: The phytochemical examination of J. curcas latex identified the presence of tannins, flavonoids, alkaloids, glycosides, sterols, saponins, and terpenes. The latex exhibited considerable antibacterial efficacy against all tested species, with inhibition zones measuring from 23 mm (K. pneumoniae) to 31.3 mm (S. aureus). The MIC values varied from 6.25 mg/mL (eg, E. coli, K. pneumoniae, C. albicans) to 25 mg/mL (S. aureus, P. aeruginosa). The comparative research indicated that J. curcas latex surpassed tetracycline and had equivalent or better efficacy to ofloxacin and fluconazole against several isolates. Conclusion: The latex of J. curcas has extensive antibacterial action against major burn wound pathogens. Its efficacy can be linked to the presence of several bioactive substances. These findings point to its potential use as a topical antibacterial agent in burn wound treatment, particularly in countries with significant antibiotic resistance.

ABSTRACTChronic, non-healing wounds are a major complication of diabetes associated with high morbidity and health care expenditures estimated at $9-13 billion annually in the US. Though microbial infection and critical colonization is hypothesized to impair healing and contribute to severe outcomes such as amputation, antimicrobial therapy is inefficacious and the role of microbes in tissue repair, regeneration, and healing remains unclear. Here, in a longitudinal prospective cohort study of 100 subjects with non-infected neuropathic diabetic foot ulcer (DFU), we performed metagenomic shotgun sequencing to elucidate microbial temporal dynamics at strain-level resolution, to investigate pathogenicity and virulence of the DFU microbiome with respect to outcomes, and to determine the influence of therapeutic intervention on the DFU microbiota. Slow healing DFUs were associated with signatures of biofilm formation, host invasion, and virulence. Though antibiotic resistance was widespre...

The study of biological invasions of ecological systems has much to offer research on withinhost systems, particularly for understanding infections and developing therapies using biological agents. Thanks to the groundwork established in other fields, such as community ecology and evolutionary biology, and to modern methods of measurement and quantification, the study of microbiomes has quickly become a field at the forefront of modern systems biology. Investigations of host-associated microbiomes (e.g., for studying human health) are often centered on measuring and explaining the structure, functions and stability of these communities. This momentum promises to rapidly advance our understanding of ecological networks and their stability, resilience and resistance to invasions. However, intrinsic properties of host-associated microbiomes that differ from those of free-living systems present challenges to the development of a within-host invasion ecology framework. The elucidation of principles underlying the invasibility of within-host networks will ultimately help in the development of medical applications and help shape our understanding of human health and disease.

Microbes produce a biofilm matrix consisting of proteins, extracellular DNA, and polysaccharides that is integral in the formation of bacterial communities. Historical studies of polysaccharides revealed that their overproduction often alters the colony morphology and can be diagnostic in identifying certain species. The polysaccharide component of the matrix can provide many diverse benefits to the cells in the biofilm, including adhesion, protection, and structure. Aggregative polysaccharides act as molecular glue, allowing the bacterial cells to adhere to each other as well as surfaces. Adhesion facilitates the colonization of both biotic and abiotic surfaces by allowing the bacteria to resist physical stresses imposed by fluid movement that could separate the cells from a nutrient source. Polysaccharides can also provide protection from a wide range of stresses, such as desiccation, immune effectors, and predators such as phagocytic cells and amoebae. Finally, polysaccharides ca...

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The study of biological invasions of ecological systems has much to offer research on withinhost systems, particularly for understanding infections and developing therapies using biological agents. Thanks to the groundwork established in other fields, such as community ecology and evolutionary biology, and to modern methods of measurement and quantification, the study of microbiomes has quickly become a field at the forefront of modern systems biology. Investigations of host-associated microbiomes (e.g., for studying human health) are often centered on measuring and explaining the structure, functions and stability of these communities. This momentum promises to rapidly advance our understanding of ecological networks and their stability, resilience and resistance to invasions. However, intrinsic properties of host-associated microbiomes that differ from those of free-living systems present challenges to the development of a within-host invasion ecology framework. The elucidation of principles underlying the invasibility of within-host networks will ultimately help in the development of medical applications and help shape our understanding of human health and disease.

Bacterial biofilms are communities of microorganisms residing within a polysaccharide matrix. Dental plaque, slimy coating in tanks, and algal mats on bodies of water are examples of biofilms. It is well accepted that biofilms play important role in bacterial persistence and antibiotic resistance in chronic infections. It is acknowledged that the majority of microbial cells on earth are living in distinct communities as biofilms. In fact, it is now known that 99% of all bacteria exist in biofilms, with only 1% living in the planktonic state. It has been estimated that 65% of microbial infections are associated with biofilms which constitute a microbial multicellular lifestyle and are defined as organized communities of bacteria. The structural nature of biofilms can protect the cells against antimicrobial agents and the host's defense. The microbial community inside a biofilm can also be protected against adverse conditions such as desiccation, osmotic shock, UV radiation, or exposure to toxic compounds, or predators. Due to the heterogeneous nature of biofilms, it is likely that multiple mechanisms of antimicrobial resistance can occur.

Bacterial cells in the natural environment and in infections are rarely found in a planktonic state. They are instead arranged in well-organized communities embedded in self-produced extracellular polymeric substances (EPS) called biofilms. The biofilm lifestyle confers a wide range of properties to the residing cells that allow efficient social interaction, permit nutrient availability, and ensure optimum usage of available enzymes and resources. The biofilm structure also permits a high level of tolerance to antimicrobials and host defense mechanisms. This creates a clinical milestone in treatment of biofilm-related infections. The medical consequences of biofilm formation and associated device-related infections (DRI) have been amplified with the widespread use of implanted medical devices. However, the biofilm structure itself represents a promising target in the development of novel antibacterial drugs. Therefore, this review represents an overview on the biofilm properties and the role of the EPS in the biofilm ecosystem. In addition, it emphasises the involvement of the biofilm structure as a therapeutic target in the development of novel antimicrobials and treatment of biofilm-related infections.

Bacterial cells in the natural environment and in infections are rarely found in a planktonic state. They are instead arranged in well-organized communities embedded in self-produced extracellular polymeric substances (EPS) called biofilms. The biofilm lifestyle confers a wide range of properties to the residing cells that allow efficient social interaction, permit nutrient availability, and ensure optimum usage of available enzymes and resources. The biofilm structure also permits a high level of tolerance to antimicrobials and host defense mechanisms. This creates a clinical milestone in treatment of biofilm-related infections. The medical consequences of biofilm formation and associated device-related infections (DRI) have been amplified with the widespread use of implanted medical devices. However, the biofilm structure itself represents a promising target in the development of novel antibacterial drugs. Therefore, this review represents an overview on the biofilm properties and the role of the EPS in the biofilm ecosystem. In addition, it emphasises the involvement of the biofilm structure as a therapeutic target in the development of novel antimicrobials and treatment of biofilm-related infections.

Intercellular communication is a crucial process for the multicellular community in both prokaryotes and eukaryotes. Indole has been recognized as a new member of the signal molecules which enables the regulated control of various bacterial phenotypes. To elucidate the interspecies relationship among enteric microorganisms via indole signaling, Klebsiella pneumoniae (KP) culture was treated with indole solution and examined for the pathogenicity by using various phenotypic tests. Both synthetic and naturally-produced indole preparations had no deteriorating effect on growth and autoaggregative capacity of KP. The results showed that biofilm formation of carbapenem-susceptible K. pneumoniae (KP-S) was clearly induced by indole exposure (& 2-10 folds), whereas no significant difference was observed in the resistant counterpart. In addition, the tolerance to b-lactam antibiotics of KP was altered upon exposure to indole and/ or derivatives assessed by Kirby-Bauer disk diffusion test. Taken together, our finding indicates the functional role of indole in changing or modulating pathogenic behaviors of other bacteria.

Biofilm is a community of microorganisms embedded in extracellular polymeric substances. Organisms in their biofilm form get many advantages over their planktonic counterparts, making them prefer to exist as biofilm. Biofilm formation involves adhesion of microbes on surfaces, followed by maturation stage which is controlled by quorum sensing (QS). Organisms present in biofilm can be 10-1000 times more resistant to antimicrobials compared to their planktonic stage. This may be due to incomplete penetration of antibiotics into the biofilm, slow growth rate of organisms in biofilm, or certain phenotypic changes. Biofilm forming ability of microorganisms has been a source of problems for human health, industry, and agriculture. Natural products including plant extracts, and quorum sensing inhibitors seem to be good alternatives of conventional antibiotics against biofilms. Biofilm forming microbes can be exploited for beneficial applications viz. waste water treatment, N 2 fixation, oil degradation, and heavy metal sequestration. This review describes salient features of microbial biofilms, quorum sensing and drugresistance in biofilms, problems caused by biofilms, their potential applications, and methods available for their study.

Microbes typically exist in mixed communities and display complex synergistic and antagonistic interactions. The Type VI secretion system (T6SS) is widespread in Gram-negative bacteria and represents a contractile nano-machine that can fire effector proteins directly into neighbouring cells. The primary role assigned to the T6SS is to function as a potent weapon during inter-bacterial competition, delivering antibacterial effectors into rival bacterial cells. However, it has recently emerged that the T6SS can also be used as a powerful weapon against fungal competitors, and the first fungal-specific T6SS effector proteins, Tfe1 and Tfe2, have been identified. These effectors act via distinct mechanisms against a variety of fungal species to cause cell death. Tfe1 intoxication triggers plasma membrane depolarisation, whilst Tfe2 disrupts nutrient uptake and induces autophagy. Based on the frequent coexistence of bacteria and fungi in microbial communities, we propose that T6SS-depend...

Even though polymicrobial infections are very common, their study is still at an early stage. In these infections, particularly when biofilms are formed, microorganisms establish a complex network of interactions that play an important role in the severity and progression of disease, with consequences in virulence, in the immune response and in the effectiveness of antimicrobial therapy. In an era in which resistance to antimicrobials is an alarming and rapidly expanding scenario, the choice of appropriate treatment is paramount, with consequences in preventing the development and spreading of resistances. The biotic interactions between strains of Staphylococcus aureus and Enterococcus faecalis in the presence of cephalexin were studied, both in the planktonic state and in biofilms. The strains were selected from a collection of veterinary clinical isolates. The studies were conducted for single-species or dual-species combinations and at two time points: 6h and 20 h. The viability of cells in the planktonic phase was determined by culture-dependent methods and the cellular viability of biofilms was evaluated through metabolic activity quantification and Live/Dead staining. The biofilm-forming capacity of the tested strains was quantified, as well as their matrix production. Results & Conclusions We showed that co-culturing the two strains in the presence of cephalexin benefits the most sensitive (S. aureus) in a greater extent than the resistant one (E. faecalis). In fact S. aureus were able to survive and were culturable when coexposed to concentrations of cephalexin higher than their minimum inhibitory concentration. The biofilm-forming capacity of the most sensitive strain is affected in the same manner, however at an early stage of biofilm formation both strains benefit from being in co-culture. Exposure to cephalexin caused changes in the morphology of the strains.

The respiratory tract is constantly exposed to the environment and displays a favorable niche for colonizing microorganisms. However, the effects of respiratory bacterial carriage on the immune system and its implications for secondary responses remain largely unclear. We have employed respiratory carriage with as the underlying model to comprehensively address effects on subsequent immune responses. Carriage was associated with the stimulation of -specific CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ T cell responses, and broad transcriptional activation was observed in CD4⁺CD25⁺ T cells. Importantly, transfer of leukocytes from carriers to acutely infected mice, resulted in a significantly increased bacterial burden in the recipient's upper respiratory tract. In contrast, we found that respiratory carriage resulted in a significant benefit for the host in systemic infection with Adaptive responses to vaccination and influenza A virus infection, were unaffected by carriage. These data showe...

Purpose: While some microorganisms, such as Staphylococcus aureus, are clearly implicated in causing tissue damage in diabetic foot ulcers (DFUs), our knowledge of the contribution of the entire microbiome to clinical outcomes is limited. We profiled the microbiome of a longitudinal sample series of 28 people with diabetes and DFUs of the heel in an attempt to better characterise the relationship between healing, infection and the microbiome. Methodology: 237 samples were analysed from 28 DFUs, collected at fortnightly intervals for six months or until healing. Microbiome profiles were generated by 16S rRNA analysis, supplemented by targeted nanopore sequencing. Results/Key findings: DFUs which failed to heal during the study period (20/28, 71.4%) were more likely to be persistently colonised with a heterogeneous community of microorganisms including anaerobes and Enterobacteriaceae (log-likelihood ratio 9.56, p=0.008). During clinically apparent infection, a reduction in the diversity of microorganisms in a DFU was often observed due to expansion of one or two taxa, with recovery in diversity at resolution. Modelling of the predicted species interactions in a single DFU with high diversity indicated that networks of metabolic interactions may exist that contribute to the formation of stable communities. Conclusion: Longitudinal profiling is an essential tool for improving our understanding of the microbiology of chronic wounds, as community dynamics associated with clinical events can only be identified by examining changes over multiple time points. The development of complex communities, particularly involving Enterobacteriaceae and strict anaerobes, may be contributing to poor outcomes in DFUs and requires further investigation.

Biofilm is a community of microorganisms embedded in extracellular polymeric substances. Organisms in their biofilm form get many advantages over their planktonic counterparts, making them prefer to exist as biofilm. Biofilm formation involves adhesion of microbes on surfaces, followed by maturation stage which is controlled by quorum sensing (QS). Organisms present in biofilm can be 10-1000 times more resistant to antimicrobials compared to their planktonic stage. This may be due to incomplete penetration of antibiotics into the biofilm, slow growth rate of organisms in biofilm, or certain phenotypic changes. Biofilm forming ability of microorganisms has been a source of problems for human health, industry, and agriculture. Natural products including plant extracts, and quorum sensing inhibitors seem to be good alternatives of conventional antibiotics against biofilms. Biofilm forming microbes can be exploited for beneficial applications viz. waste water treatment, N 2 fixation, oil degradation, and heavy metal sequestration. This review describes salient features of microbial biofilms, quorum sensing and drugresistance in biofilms, problems caused by biofilms, their potential applications, and methods available for their study.

Catheter-related urinary tract infections are one of the most common biofilm-associated diseases. Inside biofilms, bacteria cooperate, compete, or have neutral interactions. The aim was to study the interactions inside polymicrobial biofilms formed by Klebsiella pneumoniae and Enterococcus faecalis, two of the most common uropathogens. Although K. pneumoniae was the most adherent strain, it was unable to maintain dominance in the polymicrobial biofilm due to the lactic acid produced by E. faecalis in a glucose-enriched medium. This result was supported using the E. faecalis V583 ldh-1/ ldh-2 double mutant, which not inhibited the growth of K. pneumoniae since this mutant does not produce lactic acid. Lyophilized cell-free supernatants (L-CFS) obtained from E. faecalis biofilms also showed antimicrobial/antibiofilm activity against K. pneumoniae. Conversely, there were no significant differences in planktonic polymicrobial cultures. In conclusion, E. faecalis modifies the pH by lactic acid production in polymicrobial biofilms, compromising the growth of K. pneumoniae.

Antimicrobial resistance is becoming one the most serious health threats worldwide, as it not only hampers effective treatment of infectious diseases using current antibiotics, but also increases the risks of medical procedures like surgery, transplantation, bone and dental implantation, chemotherapy, or chronic wound management. To date, there are no effective measures to tackle life-threatening nosocomial infections caused by multidrug resistant bacterial species, of which Gram-negative species within the so-called "ESKAPE" pathogens are the most worrisome. Many such bacteria are frequently isolated from severely infected skin lesions such as diabetic foot ulcers (DFU). In this connection, we are pursuing new peptide constructs encompassing antimicrobial and collagenesis-inducing motifs, to tackle skin and soft tissue infections by exerting a dual effect: antimicrobial protection and faster healing of the wound. This produced peptide 3.1-PP4 showed MIC values as low as 1.0 and 2.1 µM against Escherichia coli and Pseudomonas aeruginosa, respectively, and low toxicity to HFF-1 human fibroblasts. Remarkably, the peptide was also potent against multidrug-resistant isolates of Klebsiella pneumoniae, E. coli, and P. aeruginosa (MIC values between 0.5 and 4.1 µM), and hampered the formation of/disaggregated K. pneumoniae biofilms of resistant clinical isolates. Moreover, this notable hybrid peptide retained the collagenesis-inducing behavior of the reference cosmeceutical peptide C 16-PP4 ("Matrixyl"). In conclusion, 3.1-PP4 is a highly promising lead toward development of a topical treatment for severely infected skin injuries.

Introduction Bacterial biofilms are a major worldwide healthcare problem urinary tract infections and are associated with decreasing quality of life and significant patient morbidity 1. Bacteria in biofilm mode protect themselves from antibiotics and the patient s immune system 2. Treatment of

The antibiofilm activity of a gH625 analogue was investigated to determine the in vitro inhibition and eradication of a dual-species biofilm of Candida albicans and Klebsiella pneumoniae, two leading opportunistic pathogens responsible for several resistant infections. The possibility of effectively exploiting this peptide as an alternative anti-biofilm strategy in vivo was assessed by the investigation of its efficacy on the Galleria mellonella larvae model. Results on larvae survival demonstrate a prophylactic efficacy of the peptide towards the infection of each single microorganism but mainly towards the co-infection. The expression of biofilm-related genes in vivo showed a possible synergy in virulence when these two species co-exist in the host, which was effectively prevented by the peptide. These findings provide novel insights into the treatment of medically relevant bacterial–fungal interaction.

Efficient antibiotics are being exhausted, which compromises the treatment of infections, including complicated skin and skin structure infections (cSSTI) often associated with multidrug resistant (MDR) bacteria, methicillin-resistant S. aureus (MRSA) being the most prevalent. Antimicrobial peptides (AMP) are being increasingly regarded as the new hope for the post-antibiotic era. Thus, future management of cSSTI may include use of peptides that, on the one hand, behave as AMP and, on the other, are able to promote fast and correct skin rebuilding. As such, we combined the well-known cosmeceutical pentapeptide-4 (PP4), devoid of antimicrobial action but possessing collagenesis-boosting properties, with the AMP 3.1, to afford the chimeric peptide PP4-3.1. We further produced its N-methyl imidazole derivative, MeIm-PP4-3.1. Both peptide-based constructs were evaluated in vitro against Gram-negative bacteria, Gram-positive bacteria, and Candida spp. fungi. Additionally, the antibiofilm...

The emergence of carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa raises fears of untreatable infections and poses the greatest health threats. Antimicrobial peptides (AMPs) are regarded as the most ideal solution to this menace. In this study, a set of peptides was designed based on our previously reported peptide cathelicidin-BF-15, and the lead peptide ZY4, a cyclic peptide stabilized by a disulfide bridge with high stability in vivo (the half-life is 1.8 h), showed excellent activity against P. aeruginosa and A. baumannii, including standard and clinical multidrug-resistant (MDR) strains. ZY4 killed bacteria by permeabilizing the bacterial membrane and showed low propensity to induce resistance, exhibited biofilm inhibition and eradication activities, and also killed persister cells. Notably, administration of ZY4 decreased susceptibility to lung infection by P. aeruginosa and suppressed dissemination of P. aeruginosa and A. baumannii to target organs in a...

Background: Pseudomonas aeruginosa is considered as a major cause of hospital-acquired infections due to its high antibacterial resistance. Biofilm formation is a well-known pathogenic mechanism in P. aeruginosa infections, since sessile bacteria are protected in an extracellular matrix of exopolysaccharide. The expression of polysaccharide synthesis locus (pslA gene) can be important for biofilm formation by P. aeruginosa. Objectives: The purpose of this research was to evaluate the antibiotic resistance pattern and distribution of the pslA gene among biofilmproducing P. aeruginosa isolates obtained from waste water of Burn Centre in Guilan, Iran. Materials and Methods: Fifty isolates of P. aeruginosa were obtained from waste water of a burn center. The P. aeruginosa isolates were identified using standard bacteriological procedures. Drug susceptibility test was performed by disk diffusion method for all the isolates against nine antimicrobial agents. Biofilm formation was measured by microtiter plate assay. Polymerase chain reaction (PCR) was used to identify the presence of the pslA gene among the isolates. Results: Biofilm formation was observed in 70% of the P. aeruginosa isolates. The potential formation of biofilm was significantly associated with resistance to gentamicin, imipenem, tobramycin and piperacillin. In addition, the pslA gene only existed in biofilm-producing isolates with a frequency of 42.9% (n = 15). The findings of the present study well demonstrated that the P. aeruginosa biofilm-producing isolates were more resistant to the tested antibiotics. Furthermore, because of wide distribution, it seems that the pslA gene is associated with biofilm formation.

Biofilms are communities of aggregated bacterial cells embedded in a self-produced extracellular polymeric matrix. Biofilms are recalcitrant to antibiotic treatment and immune defenses and are implicated in many chronic bacterial and fungal infections. In this review, we provide an overview of the contribution of biofilms to persistent infections resistant to antibiotic treatment, the impact of multispecies biofilms on drug resistance and tolerance, and recent advances in the development of antibiofilm agents. Understanding the mechanisms of antibiotic resistance and tolerance in biofilms is essential for developing new preventive and therapeutic strategies and curbing drug resistance.