Modulating biofilm can potentiate activity of novel plastic-degrading enzymes.

Plastic pollution is an increasing global issue desperately requiring a solution. Only 9% of all plastic waste has been recycled, and whilst recycling gives a second life to plastic, it is costly and there are limited downstream uses of recycled plastic, therefore an alternative is urgently needed. Biodegradation of plastic by microorganisms is a developing field of interest with the potential for bioreactors to be used alongside recycling to degrade plastic that may otherwise be sent to landfill. Here, we have identified two novel polyethylene terephthalate (PET) degrading enzymes through genomic mining and characterised their activity, including their ability to degrade PET. One of the main roadblocks facing the development of microbial enzymes as a plastic biodegradation solution, is that their efficiency is too low to facilitate development as bioremediation tools. In an innovative approach to tackle this roadblock, we hypothesised that enhancing a bacteria's ability to attach to and form a biofilm on plastic could maximise the local concentration of the enzyme around the target substrate, therefore increasing the overall rate of plastic degradation. We found that increasing biofilm levels, by manipulating the levels of the second messenger, Cyclic-di-GMP, led to increased levels of polyester degradation in cells expressing novel and well characterised polyester-degrading enzymes. This indicates that modulating biofilm formation is a viable mechanism to fast track the development of bacterial plastic bioremediation solutions.

Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria.

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton's reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis.

Critical Gram-negative pathogens, like Pseudomonas, Stenotrophomonas and Burkholderia, have become resistant to most antibiotics. Complex resistance profiles together with synergistic interactions between these organisms increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multi-drug resistant Pseudomonas aeruginosa to ß-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant pathogen Stenotrophomonas maltophilia. The same approach also inhibits cross-protection between resistant S. maltophilia and susceptible P. aeruginosa, allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections.

Enrichment of native plastic-associated biofilm communities to enhance polyester degrading activity.

Plastic pollution is an increasing worldwide problem urgently requiring a solution. While recycling rates are increasing globally, only 9% of all plastic waste has been recycled, and with the cost and limited downstream uses of recycled plastic, an alternative is needed. Here, we found that expanded polystyrene (EPS) promoted high levels of bacterial biofilm formation and sought out environmental EPS waste to characterize these native communities. We demonstrated that the EPS attached communities had limited plastic degrading activity. We then performed a long-term enrichment experiment where we placed a robust selection pressure on these communities by limiting carbon availability such that the waste plastic was the only carbon source. Seven of the resulting enriched bacterial communities had increased plastic degrading activity compared to the starting bacterial communities. Pseudomonas stutzeri was predominantly identified in six of the seven enriched communities as the strongest polyester degrader. Sequencing of one isolate of P. stutzeri revealed two putative polyesterases and one putative MHETase. This indicates that waste plastic-associated biofilms are a source for bacteria that have plastic-degrading potential, and that this potential can be unlocked through selective pressure and further in vitro enrichment experiments, resulting in biodegradative communities that are better than nature.

Understanding the Consumption of Antimicrobial Resistance-Related Content on Social Media: Twitter Analysis.

BACKGROUND: Antimicrobial resistance (AMR) is one of the most pressing concerns in our society. Today, social media can function as an important channel to disseminate information about AMR. The way in which this information is engaged with depends on a number of factors, including the target audience and the content of the social media post. OBJECTIVE: The aim of this study is to better understand how AMR-related content is consumed on the social media platform Twitter and to understand some of the drivers of engagement. This is essential to designing effective public health strategies, raising awareness about antimicrobial stewardship, and enabling academics to effectively promote their research on social media. METHODS: We took advantage of unrestricted access to the metrics associated with the Twitter bot @AntibioticResis, which has over 13,900 followers. This bot posts the latest AMR research in the format of a title and a URL link to the PubMed page for an article. The tweets do not contain other attributes such as author, affiliation, or journal. Therefore, engagement with the tweets is only affected by the words used in the titles. Using negative binomial regression models, we measured the impact of pathogen names in paper titles, academic attention inferred from publication counts, and general attention estimated from Twitter on URL clicks to AMR research papers. RESULTS: Followers of @AntibioticResis consisted primarily of health care professionals and academic researchers whose interests comprised mainly AMR, infectious diseases, microbiology, and public health. Three World Health Organization (WHO) critical priority pathogens-Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae-were positively associated with URL clicks. Papers with shorter titles tended to have more engagements. We also described some key linguistic characteristics that should be considered when a researcher is trying to maximize engagement with their publication. CONCLUSIONS: Our finding suggests that specific pathogens gain more attention on Twitter than others and that the levels of attention do not necessarily correspond to their status on the WHO priority pathogen list. This suggests that more targeted public health strategies may be needed to raise awareness about AMR among specific pathogens. Analysis of follower data suggests that in the busy schedules of health care professionals, social media offers a fast and accessible gateway to staying abreast of the latest developments in this field.

Using the Galleria mellonella burn wound and infection model to identify and characterize potential wound probiotics.

Burn wound infection is the leading cause of mortality among burn wound patients. One of the most commonly isolated bacterial burn wound pathogens is Pseudomonas aeruginosa, a notorious nosocomial multidrug-resistant pathogen. As a consequence of its recalcitrance to frontline antibiotic therapy, there is an urgent need to develop alternative treatment avenues to tackle this pathogen. One potential alternative infection prevention measure is to seed the wound bed with probiotic bacteria. Several species of Lactobacillus, a common commensal bacterium, have been previously reported to display growth inhibition activity against wound pathogens. Various species of this genus have also been shown to augment the wound healing process, which makes it a promising potential therapeutic agent. Due to the complexity of the burn wound trauma and burn wound infection, an in vivo model is required for the development of novel therapeutics. There are multiple in vivo models that are currently available, the most common among them being the murine model. However, mammalian burn wound infection models are logistically challenging, do not lend themselves to screening approaches and come with significant concerns around ethics and animal welfare. Recently, an invertebrate burn wound and infection model using G. mellonella has been established. This model addresses several of the challenges of more advanced animal models, such as affordability, maintenance and reduced ethical concerns. This study validates the capacity of this model to screen for potential wound probiotics by demonstrating that a variety of Lactobacillus spp. can limit P. aeruginosa burn wound infection and improve survival.

Furanone loaded aerogels are effective antibiofilm therapeutics in a model of chronic Pseudomonas aeruginosa wound infection.

Almost 80% of chronic wounds have a bacterial biofilm present. These wound biofilms are caused by a range of organisms and are often polymicrobial. Pseudomonas aeruginosa is one of the most common causative organisms in wound infections and readily forms biofilms in wounds. To coordinate this, P. aeruginosa uses a process known as quorum sensing. Structural homologues of the quorum sensing signalling molecules have been used to disrupt this communication and prevent biofilm formation by Pseudomonas. However, these compounds have not yet reached clinical use. Here, we report the production and characterisation of a lyophilised PVA aerogel for use in delivering furanones to wound biofilms. PVA aerogels successfully release a model antimicrobial and two naturally occurring furanones in an aqueous environment. Furanone loaded aerogels inhibited biofilm formation in P. aeruginosa by up to 98.80%. Further, furanone loaded aerogels successfully reduced total biomass of preformed biofilms. Treatment with a sotolon loaded aerogel yielded a 5.16 log reduction in viable biofilm bound cells in a novel model of chronic wound biofilm, equivalent to the current wound therapy Aquacel AG. These results highlight the potential utility of aerogels in drug delivery to infected wounds and supports the use of biofilm inhibitory compounds as wound therapeutics.

Artificial sweeteners inhibit multidrug-resistant pathogen growth and potentiate antibiotic activity.

Antimicrobial resistance is one of the most pressing concerns of our time. The human diet is rich with compounds that alter bacterial gut communities and virulence-associated behaviours, suggesting food additives may be a niche for the discovery of novel anti-virulence compounds. Here, we identify three artificial sweeteners, saccharin, cyclamate and acesulfame-K (ace-K), that have a major growth inhibitory effect on priority pathogens. We further characterise the impact of ace-K on multidrug-resistant Acinetobacter baumannii, demonstrating that it can disable virulence behaviours such as biofilm formation, motility and the ability to acquire exogenous antibiotic-resistant genes. Further analysis revealed the mechanism of growth inhibition is through bulge-mediated cell lysis and that cells can be rescued by cation supplementation. Antibiotic sensitivity assays demonstrated that at sub-lethal concentrations, ace-K can resensitise A. baumannii to last resort antibiotics, including carbapenems. Using a novel ex vivo porcine skin wound model, we show that ace-K antimicrobial activity is maintained in the wound microenvironment. Our findings demonstrate the influence of artificial sweeteners on pathogen behaviour and uncover their therapeutic potential.

Using next generation antimicrobials to target the mechanisms of infection.

The remarkable impact of antibiotics on human health is being eroded at an alarming rate by the emergence of multidrug resistant pathogens. There is a recognised consensus that new strategies to tackle infection are urgently needed to limit the devasting impact of antibiotic resistance on our global healthcare infrastructure. Next generation antimicrobials (NGAs) are compounds that target bacterial virulence factors to disrupt pathogenic potential without impacting bacterial viability. By disabling the key virulence factors required to establish and maintain infection, NGAs make pathogens more vulnerable to clearance by the immune system and can potentially render them more susceptible to traditional antibiotics. In this review, we discuss the developing field of NGAs and how advancements in this area could offer a viable standalone alternative to traditional antibiotics or an effective means to prolong antibiotic efficacy when used in combination.

A high-efficiency scar-free genome-editing toolkit for Acinetobacter baumannii.

BACKGROUND: The current mutagenesis tools for Acinetobacter baumannii leave selection markers or residual sequences behind, or involve tedious counterselection and screening steps. Furthermore, they are usually adapted for model strains, rather than for MDR clinical isolates. OBJECTIVES: To develop a scar-free genome-editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075. METHODS: We prove the efficiency of our adapted genome-editing system by deleting the multidrug efflux pumps craA, cmlA5 and resistance island 2 (RI2), as well as curing plasmid p1AB5075, and combining these mutations. We then characterized the susceptibility of the mutants compared with the WT to different antibiotics (i.e. chloramphenicol, amikacin and tobramycin) by disc diffusion assays and determined the MIC for each strain. RESULTS: We successfully adapted the genome-editing protocol to A. baumannii AB5075, achieving a double recombination frequency close to 100% and routinely securing the construction of a mutant within 10 working days. Furthermore, we show that both CraA and p1AB5075 are involved in chloramphenicol resistance, and that RI2 and p1AB5075 play a role in resistance to amikacin and tobramycin. CONCLUSIONS: We have developed a versatile and highly efficient genome-editing tool for A. baumannii. We have demonstrated it can be used to modify both the chromosome and native plasmids. By challenging the method, we show the role of CraA and p1AB5075 in antibiotic resistance.

Identification and characterization of novel endolysins targeting Gardnerella vaginalis biofilms to treat bacterial vaginosis.

Bacterial vaginosis (BV) is a recurrent dysbiosis that is frequently associated with preterm birth, increased risk for acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs). The overgrowth of a key pathobiont, Gardnerella vaginalis, as a recalcitrant biofilm is central to the development of this dysbiosis. Overgrowth of vaginal biofilms, seeded by initial G. vaginalis colonization, leads to recurrent symptomatic BV which is poorly resolved by classically used antibiotics. In this light, the use of bacteriophages and/or their proteins, represents a promising alternative. Here we identify 84 diverse anti-Gardnerella endolysins across 7 protein families. A subset of 36 endolysin candidates were refactored and overexpressed in an E. coli BL21 (DE3) system and 5 biochemically and structurally diverse endolysins were fully characterized. Each candidate endolysin showed good lytic activity against planktonic G. vaginalis ATCC14018, as well as G. vaginalis clinical isolates. These endolysin candidates were assayed in biofilm prevention and disruption assays, with biofilm disruption at low microgram concentrations (5 µg/ml) observed. In addition to clonal G. vaginalis biofilms, endolysin candidates could also successfully disrupt polyspecies biofilms. Importantly, none of our candidates showed lytic activity against commensal lactobacilli present in the vaginal microbiota such as L. crispatus, L. jensenii, L. gasseri, and L. iners or against Atopobium vaginae (currently classified as Fannyhessa vaginae). The potency and selectivity of these novel endolysins constitute a promising alternative treatment to combat BV, avoiding problems associated with antibiotic resistance, while retaining beneficial commensal bacteria in the vaginal flora. The diverse library of candidates reported here represents a strong repository of endolysins for further preclinical development.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse ß-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Antibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer ­ called the cell envelope ­ that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into Escherichia coli bacteria, which made the bacteria resistant to antibiotics. Furniss, Kaderabkova et al. then stopped the modified E. coli from making DsbA, which led to the antibiotic resistance proteins becoming unstable and breaking down because they could not fold correctly. Further experiments showed that blocking DsbA with a chemical inhibitor in other pathogenic species of Gram-negative bacteria made these bacteria more sensitive to antibiotics that they would normally resist. To demonstrate that using this approach could work to stop infections by these bacteria, Furniss, Kaderabkova et al. used Gram-negative bacteria that produced antibiotic resistance proteins but could not make DsbA to infect insect larvae. The larvae were then treated with antibiotics, which increased their survival rate, indicating that blocking DsbA may be a good approach to tackling antibiotic resistant bacteria. According to the World Health Organization, developing new treatments against Gram-negative bacteria is of critical importance, but the discovery of new drugs has ground to a halt. One way around this is to develop ways to make existing drugs work better. Making drugs that block DsbA could offer a way to treat resistant infections using existing antibiotics in the future.

Antibiotic Resistance Mechanisms and Their Transmission in Acinetobacter baumannii.

The discovery of penicillin over 90 years ago and its subsequent uptake by healthcare systems around the world revolutionised global health. It marked the beginning of a golden age in antibiotic discovery with new antibiotics readily discovered from natural sources and refined into therapies that saved millions of lives. Towards the end of the last century, the rate of discovery slowed to a near standstill. The lack of discovery is compounded by the rapid emergence and spread of bacterial pathogens that exhibit resistance to multiple antibiotic therapies and threaten the sustainability of global healthcare systems. Acinetobacter baumannii is an opportunistic pathogen whose prevalence and impact has grown significantly over the last 20 years. It is recognised as a barometer of the antibiotic resistance crisis due to the diverse array of mechanisms by which it can become resistant.

Burns and biofilms: priority pathogens and in vivo models.

Burn wounds can create significant damage to human skin, compromising one of the key barriers to infection. The leading cause of death among burn wound patients is infection. Even in the patients that survive, infections can be notoriously difficult to treat and can cause lasting damage, with delayed healing and prolonged hospital stays. Biofilm formation in the burn wound site is a major contributing factor to the failure of burn treatment regimens and mortality as a result of burn wound infection. Bacteria forming a biofilm or a bacterial community encased in a polysaccharide matrix are more resistant to disinfection, the rigors of the host immune system, and critically, more tolerant to antibiotics. Burn wound-associated biofilms are also thought to act as a launchpad for bacteria to establish deeper, systemic infection and ultimately bacteremia and sepsis. In this review, we discuss some of the leading burn wound pathogens and outline how they regulate biofilm formation in the burn wound microenvironment. We also discuss the new and emerging models that are available to study burn wound biofilm formation in vivo.

Evaluation of Lesions and Viral Antigen Distribution in Domestic Pigs Inoculated Intranasally with African Swine Fever Virus Ken05/Tk1 (Genotype X).

The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.

Inactivation of African Swine Fever Virus by reagents commonly used in containment laboratories.

Rapid and effective virus inactivation is an essential step for safe diagnostic testing and for research and vaccine development using infectious viruses. We characterised the reduction of African Swine Fever Virus (ASFV) infectivity using Virkon™ S (Lanxess) 1% w/v disinfectant, FACS™ Lysing buffer (BD), and AVL™ buffer (Qiagen), using porcine cell culture. No virus was detected following a 30 s 20:1 v/v mixing ratio of Virkon™ S 1% with high titre ASFV, supporting its effective use as a laboratory surface disinfectant. FACS™ Lysing and AVL™ buffers also inactivated ASFV, permitting safe removal of treated infected samples from high containment facilities.

An Invertebrate Burn Wound Model That Recapitulates the Hallmarks of Burn Trauma and Infection Seen in Mammalian Models.

The primary reason for skin graft failure and the mortality of burn wound patients, particularly those in burn intensive care centers, is bacterial infection. Several animal models exist to study burn wound pathogens. The most commonly used model is the mouse, which can be used to study virulence determinants and pathogenicity of a wide range of clinically relevant burn wound pathogens. However, animal models of burn wound pathogenicity are governed by strict ethical guidelines and hindered by high levels of animal suffering and the high level of training that is required to achieve consistent reproducible results. In this study, we describe for the first time an invertebrate model of burn trauma and concomitant wound infection. We demonstrate that this model recapitulates many of the hallmarks of burn trauma and wound infection seen in mammalian models and in human patients. We outline how this model can be used to discriminate between high and low pathogenicity strains of two of the most common burn wound colonizers Pseudomonas aeruginosa and Staphylococcus aureus, and multi-drug resistant Acinetobacter baumannii. This model is less ethically challenging than traditional vertebrate burn wound models and has the capacity to enable experiments such as high throughput screening of both anti-infective compounds and genetic mutant libraries.

Substitution of warthog NF-κB motifs into RELA of domestic pigs is not sufficient to confer resilience to African swine fever virus.

African swine fever virus (ASFV) causes a lethal, haemorrhagic disease in domestic swine that threatens pig production across the globe. Unlike domestic pigs, warthogs, which are wildlife hosts of the virus, do not succumb to the lethal effects of infection. There are three amino acid differences between the sequence of the warthog and domestic pig RELA protein; a subunit of the NF-κB transcription factor that plays a key role in regulating the immune response to infections. Domestic pigs with all 3 or 2 of the amino acids from the warthog RELA orthologue have been generated by gene editing. To assess if these variations confer resilience to ASF we established an intranasal challenge model with a moderately virulent ASFV. No difference in clinical, virological or pathological parameters were observed in domestic pigs with the 2 amino acid substitution. Domestic pigs with all 3 amino acids found in warthog RELA were not resilient to ASF but a delay in onset of clinical signs and less viral DNA in blood samples and nasal secretions was observed in some animals. Inclusion of these and additional warthog genetic traits into domestic pigs may be one way to assist in combating the devastating impact of ASFV.

Quorum Sensing Signaling Alters Virulence Potential and Population Dynamics in Complex Microbiome-Host Interactomes.

Despite the discovery of the first N-acyl homoserine lactone (AHL) based quorum sensing (QS) in the marine environment, relatively little is known about the abundance, nature and diversity of AHL QS systems in this diverse ecosystem. Establishing the prevalence and diversity of AHL QS systems and how they may influence population dynamics within the marine ecosystem, may give a greater insight into the evolution of AHLs as signaling molecules in this important and largely unexplored niche. Microbiome profiling of Stelletta normani and BD1268 sponge samples identified several potential QS active genera. Subsequent biosensor-based screening of a library of 650 marine sponge bacterial isolates identified 10 isolates that could activate at least one of three AHL biosensor strains. Each was further validated and profiled by Ultra-High Performance Liquid Chromatography Mass Spectrometry, with AHLs being detected in 8 out of 10 isolate extracts. Co-culture of QS active isolates with S. normani marine sponge samples led to the isolation of genera such as Pseudomonas and Paenibacillus, both of which were low abundance in the S. normani microbiome. Surprisingly however, addition of AHLs to isolates harvested following co-culture did not measurably affect either growth or biofilm of these strains. Addition of supernatants from QS active strains did however impact significantly on biofilm formation of the marine Bacillus sp. CH8a sporeforming strain suggesting a role for QS systems in moderating the microbe-microbe interaction in marine sponges. Genome sequencing and phylogenetic analysis of a QS positive Psychrobacter isolate identified several QS associated systems, although no classical QS synthase gene was identified. The stark contrast between the biodiverse sponge microbiome and the relatively limited diversity that was observed on standard culture media, even in the presence of QS active compounds, serves to underscore the extent of diversity that remains to be brought into culture.