Pharmaceutical Residues in Edible Oysters along the Coasts of the East and South China Seas and Associated Health Risks to Humans and Wildlife.

The investigation of pharmaceuticals as emerging contaminants in marine biota has been insufficient. In this study, we examined the presence of 51 pharmaceuticals in edible oysters along the coasts of the East and South China Seas. Only nine pharmaceuticals were detected. The mean concentrations of all measured pharmaceuticals in oysters per site ranged from 0.804 to 15.1 ng g-1 of dry weight, with antihistamines being the most common. Brompheniramine and promethazine were identified in biota samples for the first time. Although no significant health risks to humans were identified through consumption of oysters, 100-1000 times higher health risks were observed for wildlife like water birds, seasnails, and starfishes. Specifically, sea snails that primarily feed on oysters were found to be at risk of exposure to ciprofloxacin, brompheniramine, and promethazine. These high risks could be attributed to the monotonous diet habits and relatively limited food sources of these organisms. Furthermore, taking chirality into consideration, chlorpheniramine in the oysters was enriched by the S-enantiomer, with a relative potency 1.1-1.3 times higher when chlorpheniramine was considered as a racemate. Overall, this study highlights the prevalence of antihistamines in seafood and underscores the importance of studying enantioselectivities of pharmaceuticals in health risk assessments.

Juglone as a Natural Quorum Sensing Inhibitor against Pseudomonas aeruginosa pqs-Mediated Virulence and Biofilms.

Pseudomonas aeruginosa is a notorious opportunistic pathogen associated with chronic biofilm-related infections, posing a significant challenge to effective treatment strategies. Quorum sensing (QS) and biofilm formation are critical virulence factors employed by P. aeruginosa, contributing to its pathogenicity and antibiotic resistance. Other than the homoserine-based QS systems, P. aeruginosa also possesses the quinolone-based Pseudomonas quinolone signal (PQS) QS signaling. Synthesis of the PQS signaling molecule is achieved by the pqsABCDEH operon, whereas the PQS signaling response was mediated by the PqsR receptor. In this study, we report the discovery of a novel natural compound, Juglone, with potent inhibitory effects on pqs QS and biofilm formation in P. aeruginosa. Through an extensive screening of natural compounds from diverse sources, we identified Juglone, a natural compound from walnut, as a promising candidate. We showed that Juglone could inhibit PqsR and the molecular docking results revealed that Juglone could potentially bind to the PqsR active site. Furthermore, Juglone could inhibit pqs-regulated virulence factors, such as pyocyanin and the PQS QS signaling molecule. Juglone could also significantly reduce both the quantity and quality of P. aeruginosa biofilms. Notably, this compound exhibited minimal cytotoxicity toward mammalian cells, suggesting its potential safety for therapeutic applications. To explore the clinical relevance of Juglone, we investigated its combinatorial effects with colistin, a commonly used antibiotic against P. aeruginosa infections. The Juglone-colistin combinatorial treatment could eliminate biofilms formed by wild-type P. aeruginosa PAO1 and its clinical isolates collected from cystic fibrosis patients. The Juglone-colistin combinatorial therapy dramatically improved colistin efficacy and reduced inflammation in a wound infection model, indicating its potential for clinical utility. In conclusion, the discovery of Juglone provides insights into the development of innovative antivirulence therapeutic strategies to combat P. aeruginosa biofilm-associated infections.

Bacterivorous nematodes decipher microbial iron siderophores as prey cue in predator-prey interactions.

The minimal levels of biological-available iron in the environment impose growth limitation on all living organisms. Microbes often secrete high iron-binding-affinity siderophores at high concentrations for scavenging iron from the iron-limited habitats. However, the high prevalence of siderophores released by bacteria into the environment raises an intriguing question whether this chemical cue can be detected by bacterivorous predators. Here, we show that the bacterivorous Caenorhabditis elegans nematode could employ its chemosensory receptor Odr-10 to detect pyoverdine, an iron siderophore secreted by an environmental bacterium, Pseudomonas aeruginosa. This enabled the nematode predator to migrate toward the prey. Our soil microcosm study showed that the detection of pyoverdine and subsequent feeding of P. aeruginosa prey by C. elegans could lead to the expansion of its population. These results showed that siderophores are a prey chemical cue by predators, with key implications in predator-prey interactions.

Oxidative stress induced by Etoposide anti-cancer chemotherapy drives the emergence of tumor-associated bacteria resistance to fluoroquinolones.

INTRODUCTION: Antibiotic-resistant bacterial infections, such as Pseudomonas aeruginosa and Staphylococcus aureus, are prevalent in lung cancer patients, resulting in poor clinical outcomes and high mortality. Etoposide (ETO) is an FDA-approved chemotherapy drug that kills cancer cells by damaging DNA through oxidative stress. However, it is unclear if ETO can cause unintentional side effects on tumor-associated microbial pathogens, such as inducing antibiotic resistance. OBJECTIVES: We aimed to show that prolonged ETO treatment could unintendedly confer fluoroquinolone antibiotic resistance to P. aeruginosa, and evaluate the effect of tumor-associated P. aeruginosa on tumor progression. METHODS: We employed experimental evolution assay to treat P. aeruginosa with prolonged ETO exposure, evaluated the ciprofloxacin resistance, and elucidated the gene mutations by DNA sequencing. We also established a lung tumor-P. aeruginosa bacterial model to study the role of ETO-evolved intra-tumoral bacteria in tumor progression using immunostaining and confocal microscopy. RESULTS: ETO could generate oxidative stress and lead to gene mutations in P. aeruginosa, especially the gyrase (gyrA) gene, resulting in acquired fluoroquinolone resistance. We further demonstrated using a microfluidic-based lung tumor-P. aeruginosa coculture model that bacteria can evolve ciprofloxacin (CIP) resistance in a tumor microenvironment. Moreover, ETO-induced CIP-resistant (EICR) mutants could form multicellular biofilms which protected tumor cells from ETO killing and enabled tumor progression. CONCLUSION: Overall, our preclinical proof-of-concept provides insights into how anti-cancer chemotherapy could inadvertently allow tumor-associated bacteria to acquire antibiotic resistance mutations and shed new light on the development of novel anti-cancer treatments based on anti-bacterial strategies.

A Bacterial Quorum Sensing Regulated Protease Inhibits Host Immune Responses by Cleaving Death Domains of Innate Immune Adaptors.

Innate immune adaptor proteins are critical components of the innate immune system that propagate pro-inflammatory responses from their upstream receptors, and lead to pathogen clearance from the host. Bacterial pathogens have developed strategies to survive inside host cells without triggering the innate immune surveillance in ways that are still not fully understood. Here, it is reported that Pseudomonas aeruginosa induces its quorum sensing mechanism after macrophage engulfment. Further investigation of its secretome identified a quorum sensing regulated product, LasB, is responsible for innate immune suppression depending on the MyD88-mediated signaling. Moreover, it is showed that this specific type of pathogen-mediated innate immune suppression is due to the enzymatic digestion of the death domains of the innate immune adaptors, mainly MyD88, and attributed to LasB's large substrate binding groove. Lastly, it is demonstrated that the secretion of LasB from P. aeruginosa directly contributed to MyD88 degradation within macrophages. Hence, it is discovered an example of bacterial quorum sensing-regulated cellular innate immune suppression by direct cleavage of immune adaptors.

Simultaneous Dissemination of Nanoplastics and Antibiotic Resistance by Nematode Couriers.

Nanoplastics (NPs) are increasingly recognized as a newly emerging pollutant in the environment. NPs can enable the colonization of microbial pathogens on their surfaces and adsorb toxic pollutants, such as heavy metals and residual antibiotics. Although the dissemination of plastic particles in water bodies and the atmosphere is widely studied, the dissemination of NPs and adsorbed pollutants on land, via biological means, is poorly understood. Since soil animals, such as the bacterivorous nematode Caenorhabditis elegans (C. elegans), are highly mobile, this raises the possibility that they play an active role in disseminating NPs and adsorbed pollutants. Here, we established that antibiotic-resistant bacteria could aggregate with antibiotic-adsorbed NPs to form antibiotic-adsorbed NP-antibiotic resistant bacteria (ANP-ARB) aggregates, using polymyxins (colistin) as a proof-of-concept. Colistin-resistant mcr-1 bearing Escherichia coli from a mixed population of resistant and sensitive bacteria selectively aggregate with colistin-ANPs. In the soil microcosm, C. elegans fed on ANP-ARB clusters, resulting in the rapid spread of ANP-ARB by the nematodes across the soil at a rate of 40-60 cm per day. Our work revealed insights into how NPs could still disseminate across the soil faster than previously thought by "hitching a ride" in soil animals and acting as agents of antibiotic-resistant pathogens and antibiotic contaminants. This poses direct risks to ecology, agricultural sustainability, and human health.

Biofilms exacerbate atherogenesis through macrophage-induced inflammatory responses in a fibrous plaque microsystem model.

BACKGROUND: Microbes have been implicated in atherosclerosis development and progression, but the impact of bacterial-based biofilms on fibrous plaque rupture remains poorly understood. RESULTS: Here, we developed a comprehensive atherosclerotic model to reflect the progression of fibrous plaque under biofilm-induced inflammation (FP-I). High expressions of biofilm-specific biomarkers algD, pelA and pslB validated the presence of biofilms. Biofilm promotes the polarization of macrophages towards a pro-inflammatory (M1) phenotype, as demonstrated by an increase in M1 macrophage-specific marker CD80 expression in CD68+ macrophages. The increase in the number of intracellular lipid droplets (LDs) and foam cell percentage highlighted the potential role of biofilms on lipid synthesis or metabolic pathways in macrophage-derived foam cells. In addition, collagen I production by myofibroblasts associated with the fibrous cap was significantly reduced along with the promotion of apoptosis of myofibroblasts, indicating that biofilms affect the structural integrity of the fibrous cap and potentially undermine its strength. CONCLUSION: We validated the unique role of biofilm-based inflammation in exacerbating fibrous plaque damage in the FP-I model, increasing fibrous plaque instability and risk of thrombosis. Our results lay the foundation for mechanistic studies of the role of biofilms in fibrous plaques, allowing the evaluation of preclinical combination strategies for drug therapy. STATEMENT OF SIGNIFICANCE: A microsystem-based model was developed to reveal interactions in fibrous plaque during biofilm-induced inflammation (FP-I). Real-time assessment of biofilm formation and its role in fibrous plaque progression was achieved. The presence of biofilms enhanced the expression of pro-inflammatory (M1) specific marker CD80, lipid droplets, and foam cells and reduced anti-inflammatory (M2) specific marker CD206 expression. Fibrous plaque exposure to biofilm-based inflammation reduced collagen I expression and increased apoptosis marker Caspase-3 expression significantly. Overall, we demonstrate the unique role of biofilm-based inflammation in exacerbating fibrous plaque damage in the FP-I model, promoting fibrous plaque instability and enhanced thrombosis risk. Our findings lay the groundwork for mechanistic studies, facilitating the evaluation of preclinical drug combination strategies.

Distinct bacterial population dynamics and disease dissemination after biofilm dispersal and disassembly.

Microbial communities that form surface-attached biofilms must release and disperse their constituent cells into the environment to colonize fresh sites for continued survival of their species. For pathogens, biofilm dispersal is crucial for microbial transmission from environmental reservoirs to hosts, cross-host transmission, and dissemination of infections across tissues within the host. However, research on biofilm dispersal and its consequences in colonization of fresh sites remain poorly understood. Bacterial cells can depart from biofilms via stimuli-induced dispersal or disassembly due to direct degradation of the biofilm matrix, but the complex heterogeneity of bacterial populations released from biofilms rendered their study difficult. Using a novel 3D-bacterial "biofilm-dispersal-then-recolonization" (BDR) microfluidic model, we demonstrated that Pseudomonas aeruginosa biofilms undergo distinct spatiotemporal dynamics during chemical-induced dispersal (CID) and enzymatic disassembly (EDA), with contrasting consequences in recolonization and disease dissemination. Active CID required bacteria to employ bdlA dispersal gene and flagella to depart from biofilms as single cells at consistent velocities but could not recolonize fresh surfaces. This prevented the disseminated bacteria cells from infecting lung spheroids and Caenorhabditis elegans in on-chip coculture experiments. In contrast, EDA by degradation of a major biofilm exopolysaccharide (Psl) released immotile aggregates at high initial velocities, enabling the bacteria to recolonize fresh surfaces and cause infections in the hosts efficiently. Hence, biofilm dispersal is more complex than previously thought, where bacterial populations adopting distinct behavior after biofilm departure may be the key to survival of bacterial species and dissemination of diseases.

Biofilm Potentiates Cancer-Promoting Effects of Tumor-Associated Macrophages in a 3D Multi-Faceted Tumor Model.

Components of the tumor microenvironment (TME), such as tumor-associated macrophages (TAMs), influence tumor progression. The specific polarization and phenotypic transition of TAMs in the tumor microenvironment lead to two-pronged impacts that can promote or hinder cancer development and treatment. Here, a novel microfluidic multi-faceted bladder tumor model (TAMPIEB ) is developed incorporating TAMs and cancer cells to evaluate the impact of bacterial distribution on immunomodulation within the tumor microenvironment in vivo. It is demonstrated for the first time that biofilm-induced inflammatory conditions within tumors promote the transition of macrophages from a pro-inflammatory M1-like to an anti-inflammatory/pro-tumor M2-like state. Consequently, multiple roles and mechanisms by which biofilms promote cancer by inducing pro-tumor phenotypic switch of TAMs are identified, including cancer hallmarks such as reducing susceptibility to apoptosis, enhancing cell viability, and promoting epithelial-mesenchymal transition and metastasis. Furthermore, biofilms formed by extratumoral bacteria can shield tumors from immune attack by TAMs, which can be visualized through various imaging assays in situ. The study sheds light on the underlying mechanism of biofilm-mediated inflammation on tumor progression and provides new insights into combined anti-biofilm therapy and immunotherapy strategies in clinical trials.

Shotgun metagenomic sequencing analysis of ocular surface microbiome in Singapore residents with mild dry eye.

The ocular surface microbiome has implications for ocular surface inflammation and immunology. Previous shotgun metagenomics analyses were performed in China, showing results that differed according to environment and age. Patients with Sjogren's syndrome were reported to have altered conjunctival microbiome, but such studies have not been done in milder dry eye. The aim of this study is to describe the conjunctival microbiome in people with mild dry eye in Singapore. Samples were collected from 14 participants with mild dry eye and 10 age-matched comparison participants recruited from Singapore National Eye Centre (SNEC) clinics. Shotgun metagenomic sequencing analysis was employed to evaluate the conjunctival microbiome composition. Proteobacteria formed the predominant phylum in the conjunctiva. As in a study from a coastal city in China, Achromobacter spp. was numerically most abundant. Compared to age-matched controls, the conjunctival microbial composition in mild dry eye was similar. Several microorganisms, including Streptococcus spp. increased in representation with age, and the abundance of Staphylococcus correlated with Schirmer readings. In addition, when cultured corneal epithelial cells were exposed to three strains of Achromobacter xylosoxidans, cytokines such as TNF-α and IL-6 were upregulated in the cell lysates and supernatants. Ourresults suggest that age is an important factor that affects composition of the conjunctival microbiome, and relative abundance of specific microorganism may vary according to the environment of the human host.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Seasonal variation of atmospheric Pb sources in Singapore - Elemental and lead isotopic compositions of PM10 as source tracer.

Southeast Asia has become a hotspot of anthropogenic particulate matter (PM) emissions due to increased coal combustion, high-temperature industrial operations, vehicular traffic, and agricultural biomass burning. Lead (Pb), a criteria pollutant, bound to such PM can be hazardous when inhaled, even at extremely low concentrations. Precise and accurate source apportionment of atmospheric Pb is thus, critical in order to minimize its exposure. This study investigates the sources of atmospheric Pb in Singapore aerosol samples (PM10) using Pb isotopes and elemental composition as tracers of contamination sources. PM10 aerosol sampling was conducted over a period of 1 year from June 2017 to May 2018 to capture the seasonal variations in sources of atmospheric Pb. Elemental concentrations reveal particularly high enrichment factors for Pb, Cu, V, Ni and Zn, especially when under the influence of southwest (SW) and inter monsoon (IM) winds. Pb isotopic ratios across the three seasons (206/207Pb = 1.147-1.150 and 208/207Pb = 2.420-2.428) are not significantly different. The Pb isotopic signatures and V/Ni ratios for all three seasons overlap with those of gasoline, diesel and ship emissions. Moreover, V/Pb values of more than unity for SW and IM winds suggest influence of transboundary coal combustion emissions particularly from Indonesia. Consequently, using Pb isotopic fingerprints and elemental ratios, we find that the primary sources of atmospheric Pb are vehicular & ship emissions, heavy oil combustion, transboundary coal combustion emissions, waste incineration and recirculation of historic leaded gasoline.

Rapid detection of microorganisms in a fish infection microfluidics platform.

Inadequate access to clean water is detrimental to human health and aquatic industries. Waterborne pathogens can survive prolonged periods in aquatic bodies, infect commercially important seafood, and resist water disinfection, resulting in human infections. Environmental agencies and research laboratories require a relevant, portable, and cost-effective platform to monitor microbial pathogens and assess their risk of infection on a large scale. Advances in microfluidics enable better control and higher precision than traditional culture-based pathogen monitoring approaches. We demonstrated a rapid, high-throughput fish-based teleost (fish)-microbe (TelM) microfluidic-based device that simultaneously monitors waterborne pathogens in contaminated waters and assesses their infection potential under well-defined settings. A chamber-associated port allows direct access to the animal, while the transparency of the TelM platform enables clear observation of sensor readouts. As proof-of-concept, we established a wound infection model using Pseudomonas aeruginosa-contaminated water in the TelM platform, where bacteria formed biofilms on the wound and secreted a biofilm metabolite, pyoverdine. Pyoverdine was used as fluorescent sensor to correlate P. aeruginosa contamination to infection. The TelM platform was validated with environmental waterborne microbes from marine samples. Overall, the TelM platform can be readily applied to assess microbial and chemical risk in aquatic bodies in resource-constrained settings.

Biofilm matrix cloaks bacterial quorum sensing chemoattractants from predator detection.

Microbes often secrete high levels of quorum sensing (QS) autoinducers into the environment to coordinate gene expression and biofilm formation, but risk detection and subsequent predation by bacterivorous predators. With such prominent signaling molecules acting as chemoattractants that diffuse into the environment at alarmingly high concentrations, it is unclear if bacterial cells can mask their chemical trails from predator detection. Here, we describe a microbial-based anti-detection adaptation, termed as "biofilm cloak", where the biofilm prey produced biofilm matrix exopolysaccharides that "locked" and reduced the leaching of autoinducers into the milieu, thereby concealing their trails to the detection by the bacterivorous Caenorhabditis elegans nematode. The exopolysaccharides act as common good for the non-producers to hide their autoinducers from predator detection. Deficiency in chemosensory gene odr-10 in mutant animals abrogated their ability to detect autoinducers and migrate toward their prey in a directed manner, which led to lower population growth rate of animals. Hence, restriction of bacterial communication activities to the confinements of biofilms is a novel approach for predator evasion, which plays a fundamental role in shaping ecological dynamics of microbial communities and predator-prey interactions.

Biofilm dispersal induced by mechanical cutting leads to heightened foodborne pathogen dissemination.

The biofilm life cycle where bacteria alternate between biofilm and planktonic lifestyles poses major implications in food spoilage and gastrointestinal infections. Recent studies had shown that freshly biofilm-dispersed cells have a unique physiology from planktonic cells, raising the fundamental question if biofilm-dispersed cells and planktonic cells disseminate differently across food surfaces. Mechanical dislodging via cutting can cause biofilm dispersal and eventual food cross-contamination. Here, we showed that biofilm-dispersed bacteria from various foodborne pathogens were transferred from freshly cut surface at a higher rate to the cutting material than that of planktonic bacteria. When the cutting tool was used to cut a fresh surface, more biofilm-dispersed bacteria were disseminated from the cutting tool to the newly cut surface than planktonic bacteria. Our observations were applicable to cutting tools of various materials and cut surfaces, where polystyrene and surfaces with high water content were most susceptible to biofilm transfer, respectively. Simple washing with detergent and mechanical wiping could aid bacterial removal from cutting tools. Our work revealed that biofilm-dispersed cells were transferred at a higher rate than planktonic cells and cutting tool was an important medium for pathogen cross-contamination, thus providing insights in maintaining their cleanliness in food processing industries.

Improved Raman spectroscopy-based approach to assess microplastics in seafood.

Microplastics represent an emerging environmental issue and have been found almost everywhere including seafood, raising a great concern about the ecological and human health risks they pose. This study addressed the common technical challenges in the assessment of microplastics in seafood by developing an improved protocol based on Raman spectroscopy and using the green-lipped mussel Perna viridis and the Japanese jack mackerel Trachurus japonicus as the test models. Our findings identified a type of stainless-steel filter membranes with minimal Raman interference, and a combination of chemicals that achieved 99-100% digestion efficiency for both organic and inorganic biomass. This combined chemical treatment reached 90-100% recovery rates for seven types of microplastics, on which the surface modification was considered negligible and did not affect the accuracy of polymer identification based on Raman spectra, which showed 94-99% similarity to corresponding untreated microplastics. The developed extraction method for microplastics was further combined with an automated Raman mapping approach, from which our results confirmed the presence of microplastics in P. viridis and T. japonicus collected from Hong Kong waters. Identified microplastics included polypropylene, polyethylene, polystyrene and poly(ethylene terephthalate), mainly in the form of fragments and fibres. Our protocol is applicable to other biological samples, and provides an improved alternative to streamline the workflow of microplastic analysis for routine monitoring purposes.

Label-free biosensor of phagocytosis for diagnosing bacterial infections.

Phagocytic cells recognize and phagocytose invading microbes for destruction. However, bacterial pathogens can remain hidden at low levels from conventional detection or replicate intracellularly after being phagocytosed by immune cells. Current phagocytosis-detection approaches involve flow cytometry or microscopic search for rare bacteria-internalized phagocytes among large populations of uninfected cells, which poses significant challenges in research and clinical settings. Hence it is imperative to develop a rapid, non-disruptive, and label-free phagocytosis detection approach. Using deformability assays and microscopic imaging, we have demonstrated for the first time that the presence of intracellular bacteria in phagocytic blood cells led to aberrant physical properties. Specifically, human monocytes with internalized bacteria of various species were stiffer and larger compared with uninfected monocytes. Taking advantage of these physical differences, a novel microfluidics-based biosensor platform was developed to passively sort, concentrate and quantify rare monocytes with internalized pathogens (MIP) from uninfected monocyte populations for phagocytosis detection. The clinical utility of the MIP platform was demonstrated by enriching and detecting bacteria-internalized monocytes from spiked human blood samples within 1.5 h. Patient-derived clinical isolates were used to validate the utility of the MIP platform further. This proof-of-concept presents a phagocytosis detection platform that could be used to rapidly diagnose microbial infections, especially in bloodstream infections (BSIs), thereby improving the clinical outcomes for point-of-care management.

The effects of biofilms on tumor progression in a 3D cancer-biofilm microfluidic model.

Components within the tumor microenvironment, such as intratumoral bacteria (IB; within tumors), affect tumor progression. However, current experimental models have not explored the effects of extratumoral bacteria (EB; outside tumors) on cancer progression. Here, we developed a microfluidic platform to analyze the influence of bacterial distribution on bladder cancer progression under defined conditions, using uropathogenic Escherichia coli. This was achieved by establishing coating (CT) and colonizing (CL) models to simulate the different invasion and colonization modes of IB and EB in tumor tissues. We demonstrated that both EB and IB induced closer cell-cell contacts within the tumor cluster, but cancer cell viability was reduced only in the presence of IB. Interestingly, cancer stem cell counts increased significantly in the presence of EB. These outcomes were due to the formation of extracellular DNA-based biofilms by EB. Triple therapy of DNase (anti-biofilm agent), ciprofloxacin (antibiotic), and doxorubicin (anti-cancer drug) could effectively eradicate biofilms and tumors simultaneously. Our preclinical proof-of-concept provides insights on how bacteria can influence tumor progression and facilitate future research on anti-biofilm cancer management therapies.