Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms

A novel benzimidazole molecule that was identified in a small‐molecule screen and is known as antibiofilm compound 1 (ABC‐1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC‐1 was tested in various biofilm‐forming strains of S. aureus. It was demonstrated that ABC‐1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall‐associated protein dependent or cell wall‐ associated extracellular DNA (eDNA). Of note, ABC‐1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC‐1 treated strains, implying that ABC‐1 affects not only SpA but also other factors. Indeed, ABC‐1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC‐1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.     

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC–MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 ( 1 ) against S. aureus was confirmed.     

Butanol production from corncob residue using Clostridium beijerinckii NCIMB 8052

Aims: Not all lactic acid bacteria possess the ability to confer health benefits for the host. Thus, it becomes necessary to screen and characterize numerous strains to obtain ideal probiotics. Here, two Lactobacillus plantarum strains (CECT 7315 and CECT 7316) were isolated and characterized. Methods and Results: In vitro and in vivo tests were carried out for demonstrating the abilities as probiotics of CECT 7315/CECT 7316 Lact. plantarum strains. Both strains showed high ability to survive at gastro‐intestinal tract conditions and to adhere to intestinal epithelial cells, as well as great inhibitory activity against a wide range of enteropathogens and ability to induce the production of anti‐inflammatory cytokine IL‐10. Conclusions: Lactobacillus plantarum CECT 7315/CECT 7316 because of their potential probiotic properties could be excellent candidates for being tested in clinical trials aimed to demonstrate beneficial effects on human health. Significance and Impact of the Study: Probiotics are live micro‐organisms that confer a health benefit for the host. However, not all the lactic acid bacteria possess the ability to confer health benefits for the host. In this study, two Lact. plantarum strains (CECT 7315 and CECT 7316) were isolated and characterized to demonstrate their excellent qualities as potential probiotic strains.     

Biofilm formation and proteolytic activities of Pseudoalteromonas bacteria that were isolated from fish farm sediments

In order to save natural resources and supply good fishes, it is important to improve fish‐farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish‐farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture‐based methods for the identification and characterization of biofilm‐forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm‐dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.     

Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter‐diffusion geometry

The link between nitritation success in a membrane‐aerated biofilm reactor (MABR) and the composition of the initial ammonia‐ and nitrite‐oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat‐sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO₂^‐‐N/NH₄⁺‐N) were obtained in the Inoculum B‐ (55.2–56.4%) versus the Inoculum A‐ (20.2–22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100–150 µm), but the AOB profiles [based on 16S rRNA gene targeted real‐time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B‐ versus A‐initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A‐ and B‐initiated reactors were Nitrosospira spp. (48.9–61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8–63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell^?1 h^?1 versus 0.79 fmol cell^?1 h^?1). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.     

Characterization of co-culture of Aeromonas and Pseudomonas bacterial biofilm and spoilage potential on refrigerated grass carp (Ctenopharyngodon idellus)

Aeromonas and Pseudomonas are important bacterial species involved in spoilage of refrigerated freshwater fish. In this study, 10 Aeromonas and seven Pseudomonas bacterial strains were isolated from spoiled grass carp and identified. Twelve of seventeen bacterial strains showed high potential of biofilm formation and 14 of 17 can produce extracellular protease. In order to explore the spoilage capacity of dual-species, the sterile grass carp fillets were inoculated with mono- and dual-species of Aeromonas salmonicida and Pseudomonas azotoformans strains. The results revealed significantly higher levels of the total viable count and total volatile basic nitrogen in dual-species as compared to mono-species from day 6. The higher contents of histamine, cadaverine and serious degradation in muscles tissue were also observed in dual-species after 10 days of storage. Results of in vitro experiments showed that the co-culture of A. salmonicida and P. azotoformans significantly increased the bacterial maximum growth rate, promoted the biofilm formation and improved the spoilage capacity of bacterial strains. This study has revealed that the co-culture of Aeromonas and Pseudomonas bacterial strains accelerated spoilage process of grass carp and increased biofilm formation. It indicates that the mixed-cultures of spoilage micro-organisms pose a huge threat to food industry.     

Probiotic Lactobacilli Interfere with <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilm Formation In Vitro

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.     

Rapid depletion of dissolved oxygen in 96‐well microtiter plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration

Biofilm‐related research using 96‐well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96‐well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96‐well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96‐well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model. Biotechnol. Bioeng. 2009;103: 1042–1047. © 2009 Wiley Periodicals, Inc.     

Comparing isogenic strains of Beijing genotype Mycobacterium tuberculosis after acquisition of Isoniazid resistance: A proteomics approach

We determined differences in the protein abundance among two isogenic strains of Mycobacterium tuberculosis (Mtb) with different Isoniazid (INH) susceptibility profiles. The strains were isolated from a pulmonary tuberculosis patient before and after drug treatment. LC‐MS/MS analysis identified 46 Mtb proteins with altered abundance after INH resistance acquisition. Protein abundance comparisons were done evaluating the different bacterial cellular fractions (membrane, cytosol, cell wall and secreted proteins). MS data have been deposited to the ProteomeXchange with identifier PXD002986.     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Aerobic biodegradation of methyl tert-butyl ether (MTBE) by pure bacterial cultures isolated from contaminated soil

Summary Methyl tert-Butyl Ether (MTBE) has been used in gasoline as a substitute for lead-based additives, which have been demonstrated to be toxic. MTBE however, is persistent in soil and water, showing high affinity for water and low affinity for soil, and has become an important contaminant. Therefore, the aim of this work was to isolate and identify soil microorganisms capable of degrading MTBE. Two samples were taken from a gasoline-contaminated soil at a service station and 59 different bacterial strains were isolated by enrichment culture with three consecutive selective transfers. Biochemical and morphological characterization of the bacterial isolates classified them into the following groups: Bacillus, Rhodococcus, Micrococcus, Aureobacterium and Proteus. Twelve strains were selected for evaluation of MTBE biodegradation depending on visual growth and biomass production of the isolates in minimal salt broth. Six strains significantly reduced MTBE concentration (22–37%) compared to an abiotic control after 5 days of incubation. Although it has been considered that MTBE is degraded mainly by cometabolism, our results demonstrate that these microorganisms are able to reduce MTBE concentration when MTBE is the sole source of carbon.     

Effect of Volatile Organic Compounds from Bacteria on Nematodes

The five studied bacterial strains could produce volatile organic compounds (VOCs) that kill nematodes. Based on their 16S rRNA sequences, these strains were identified as Pseudochrobactrum saccharolyticum, Wautersiella falsenii, Proteus hauseri, Arthrobacter nicotianae, and Achromobacter xylosoxidans. The bacterial VOCs were extracted using solid‐phase micro‐extraction (SPME) and subsequently identified by GC/MS analysis. The VOCs covered a wide range of aldehydes, ketones, alkyls, alcohols, alkenes, esters, alkynes, acids, ethers, as well as heterocyclic and phenolic compounds. Among the 53 VOCs identified, 19 candidates, produced by different bacteria, were selected to test their nematicidal activity (NA) against Caenorhabditis elegans and Meloidogyne incognita. The seven compounds with the highest NAs were acetophenone, S‐methyl thiobutyrate, dimethyl disulfide, ethyl 3,3‐dimethylacrylate, nonan‐2‐one, 1‐methoxy‐4‐methylbenzene, and butyl isovalerate. Among them, S‐methyl thiobutyrate showed a stronger NA than the commercial insecticide dimethyl disulfide. It was reported for the first time here that the five bacterial strains as well as S‐methyl thiobutyrate, ethyl 3,3‐dimethylacrylate, 1‐methoxy‐4‐methylbenzene, and butyl isovalerate possess NA. These strains and compounds might provide new insights in the search for novel nematicides.     

Selective attraction and reproductive performance of a harpacticoid copepod in a response to biofilms

The relationship between monobacterial films and the preference of harpacticoid copepods for such films was investigated using still water multiple-choice assays with natural biofilm and sterile conditions as controls. Adult Schizopera sp. were most attracted by a heterogeneous natural biofilm, followed by monospecies-biofilms of Rhodovulum sp., Vibrio proteolyticus, and Flexibacter sp. The preferred bacterial films stemmed from different phylogenetic and physiological groups. The results indicated that the harpacticoid Schizopera sp. was effectively and differentially attracted by bacterial films. Since bacteria constitute a substantial portion of the organic carbon available at the sea bottom as nutritive sources for harpacticoid copepods, we subsequently examined the influence of 9 bacterial strains and a natural biofilm as a nutrient source on the growth and reproductive performance of ontogenetic stages (nauplii and copepodids) of Schizopera sp. The food value of bacterial strains was assayed in terms of life table data that provided growth parameters. All variables were affected by the type of food offered. A diet on Rhodovulum sp. resulted in optimal growth performance of nauplii and copepodids demonstrating that bacteria can be used as a sole diet to support postembryonic development. The present study is the first to link behavioral preferences to bacterial biofilms with life history parameters when cultivating harpacticoid copepods on the same bacterial strains as the only diet. This study revealed a discrepancy between the biofilm favored (natural biofilm) and the one leading to maximal reproductive performance (monobacterial film of Rhodovulum sp. MB253) as indicated by major life table data as net reproductive rate (R_o), mean generation time (T_m), and capacity for increase (r_c).     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Chemical Composition and Antipathogenic Activity of Artemisia annua Essential Oil from Romania

The essential oil extracted by hydrodistillation from Romanian Artemisia annua aerial parts was characterized by GC/MS analysis, which allowed the identification of 94.64% of the total oil composition. The main components were camphor (17.74%), α‐pinene (9.66%), germacrene D (7.55%), 1,8‐cineole (7.24%), trans‐β‐caryophyllene (7.02%), and artemisia ketone (6.26%). The antimicrobial activity of this essential oil was evaluated by determining the following parameters: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimal fungicidal concentration (MFC), and minimal biofilm eradication concentration (MBEC). Moreover, the soluble virulence factors were quantified with different biochemical substrates incorporated in the culture media. The reference and resistant, clinical strains proved to be susceptible to the A. annua oil, with MICs ranging from 0.51 to 16.33 mg/ml. The tested essential oil also showed good antibiofilm activity, inhibiting both the initial stage of the microbial cell adhesion to the inert substratum and the preformed mature biofilm. When used at subinhibitory concentrations, the essential oil proved to inhibit the phenotypic expression of five soluble virulence factors (hemolysins, gelatinase, DNase, lipases, and lecithinases). Briefly, the present results showed that the A. annua essential oil contained antimicrobial compounds with selective activity on Gram‐positive and Gram‐negative bacterial strains as well as on yeast strains and which also interfere with the expression of cell‐associated and soluble virulence factors.     

Induction of trap formation in nematode‐trapping fungi by bacteria‐released ammonia

A total of 11 bacterial strains were assayed for bacteria‐induced trap formation in the nematode‐trapping fungus Arthrobotrys oligospora YMF1·01883 with two‐compartmented Petri dish. These strains were identified on the basis of their 16S rRNA gene sequences. Volatile organic compounds (VOCs) of eight isolates were extracted using solid‐phase micro‐extraction (SPME) and their structures were identified based on gas chromatography‐mass spectrometry (GC‐MS). At the same time, all isolates were used for quantitative measurement of ammonia by the indophenol blue method. The effects of pure commercial compounds on inducement of trap formation in A. oligospora were tested. Taken together, results demonstrated that the predominant bacterial volatile compound inducing trap formation was ammonia. Meanwhile, ammonia also played a role in other nematode‐trapping fungi, including Arthrobotrys guizhouensis YMF1·00014, producing adhesive nets; Dactylellina phymatopaga YMF1·01474, producing adhesive knobs; Dactylellina cionopaga YMF1·01472, producing adhesive columns and Drechslerella brochopaga YMF1·01829, producing constricting rings.     

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04). Received: 23 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether by newly identified soil microorganisms in a simple mineral solution

Methyl tert-butyl ether (MTBE) is a widely used fuel ether, which has become a soil and water contaminant. In this study, 12 microbial strains were isolated from gasoline-contaminated soils and selected because of their capacity to grow in MTBE. The strains were identified by 16S/ITS rDNA gene sequencing and screened for their ability to consume MTBE aerobically in a simple mineral solution. Solid phase microoextraction and gas chromatography were used to detect MTBE degradation. High levels of MTBE biodegradation were obtained using resting cells of the bacteria Achromobacter xylosoxidans MCM1/1 (78%), Enterobacter cloacae MCM2/1 (50%), and Ochrobactrum anthropi MCM5/1 (52%) and the fungus Exophiala dermatitidis MCM3/4 (14%). Our phylogenetic analysis clearly shows that bacterial MTBE biodegraders belong to the clade of Proteobacteria. For further insight, MTBE-degrader strains were profiled by denaturing gel gradient electrophoresis (DGGE) of PCR-amplified 16S rRNA gene sequences. This approach could be used to analyse microbial community dynamics in bioremediation processes.     

Proteomannans in biofilm of Helicobacter pylori ATCC 43504

Background: The human bacterial pathogen Helicobacter pylori forms biofilms. However, the constituents of the biofilm have not been extensively investigated. In this study, we analyzed the carbohydrate and protein components of biofilm formed by H. pylori strain ATCC 43504 (NCTC 11637). Materials and Methods: Development of H. pylori biofilm was analyzed using scanning electron microscopy (SEM) and quantified using crystal violet staining. The extracted extracellular polysaccharide (EPS) matrix was analyzed using GC‐MS and nuclear magnetic resonance (NMR) analyses. Proteomic profiles of biofilms were examined by SDS–PAGE while deletion mutants of upregulated biofilm proteins were constructed and characterized. Results: Formation of H. pylori biofilm is time dependent as shown by crystal violet staining assay and SEM. NMR reveals the prevalence of 1,4‐mannosyl linkages in both developing and mature biofilms. Proteomic analysis of the biofilm indicates the upregulation of neutrophil‐activating protein A (NapA) and several stress‐induced proteins. Interestingly, the isogenic mutant napA revealed a different biofilm phenotype that showed reduced aggregated colonial structure when compared to the wild type. Conclusions: This in vitro study shows that mannose‐related proteoglycans (proteomannans) are involved in the process of H. pylori biofilm formation while the presence of upregulated NapA in the biofilm implies the potency to increase adhesiveness of H. pylori biofilm. Being a complex matrix of proteins and carbohydrates, which are probably interdependent, the H. pylori biofilm could possibly offer a protective haven for the survival of this gastric bacterial pathogen in the extragastric environments.