Communication among Oral Bacteria

Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.     

Nitrite production by ammonia-oxidizing bacteria mediates chloramine decay and resistance in a mixed-species community

As water distribution centres increasingly switch to using chloramine to disinfect drinking water, it is of paramount importance to determine the interactions of chloramine with potential biological contaminants, such as bacterial biofilms, that are found in these systems. For example, ammonia-oxidizing bacteria (AOB) are known to accelerate the decay of chloramine in drinking water systems, but it is also known that organic compounds can increase the chloramine demand. This study expanded upon our previously published model to compare the decay of chloramine in response to alginate, Pseudomonas aeruginosa, Nitrosomonas europaea and a mixed-species nitrifying culture, exploring the contributions of microbial by-products, heterotrophic bacteria and AOBs to chloramine decay. Furthermore, the contribution of AOBs to biofilm stability during chloramination was investigated. The results demonstrate that the biofilm matrix or extracellular polymeric substances (EPS), represented by alginate in these experiments, as well as high concentrations of dead or inactive cells, can drive chloramine decay rather than any specific biochemical activity of P. aeruginosa cells. Alginate was shown to reduce chloramine concentrations in a dose-dependent manner at an average rate of 0.003 mg l⁻¹ h⁻¹ per mg l⁻¹ of alginate. Additionally, metabolically active AOBs mediated the decay of chloramine, which protected members of mixed-species biofilms from chloramine-mediated disinfection. Under these conditions, nitrite produced by AOBs directly reacted with chloramine to drive its decay. In contrast, biofilms of mixed-species communities that were dominated by heterotrophic bacteria due to either the absence of ammonia, or the addition of nitrification inhibitors and glucose, were highly sensitive to chloramine. These results suggest that mixed-species biofilms are protected by a combination of biofilm matrix-mediated inactivation of chloramine as well as the conversion of ammonia to nitrite through the activity of AOBs present in the community.     

Bacterial biofilms as a natural form of existence of bacteria in the environment and host organism

Advances in microscopic analysis and molecular genetics research methods promoted the acquisition of evidence that natural bacteria populations exist predominately as substrate attached biofilms. Bacteria in biofilms are able to exchange signals and display coordinated activity that is inherent to multicellular organisms. Formation of biofilm communities turned out to be one of the main survival strategies of bacteria in their ecological niche. Bacteria in attached condition in biofilm are protected from the environmental damaging factors and effects of antibacterial substances in the environment and host organism during infection. According to contemporary conception, biofilm is a continuous layer of bacterial cells that are attached to a surface and each other, and contained in a biopolymer matrix. Such bacterial communities may be composed of bacteria of one or several species, and composed of actively functioning cells as well as latent and uncultured forms. Particular attention has recently been paid to the role of biofilms in the environment and host organism. Microorganisms form biofilm on any biotic and abiotic surfaces which creates serious problems in medicine and various areas of economic activity. Currently, it is established that biofilms are one of the pathogenetic factors of chronic inflection process formation. The review presents data on ubiquity of bacteria existence as biofilms, contemporary methods of microbial community analysis, structural-functional features of bacterial biofilms. Particular attention is paid to the role of biofilm in chronic infection process formation, heightened resistance to antibiotics of bacteria in biofilms and possible mechanisms of resistance. Screening approaches for agents against biofilms in chronic infections are discussed.     

Candida albicans: Molecular interactions with Pseudomonas aeruginosa and Staphylococcus aureus

The fields of mycology and bacteriology have traditionally functioned independently of each other despite the fundamental actuality that fungi and bacteria not only co-exist but also interact within several niches. In the clinical context, these interactions commonly occur within biofilms, which can be composed of single-species communities or mixed-species populations and recent studies have shown that the properties of mixed-species populations differ from those of their individual components. The interacting bacteria and fungi can exert effects on microbial behavior, dissemination, survival, the response to antimicrobials and, ultimately, patient prognosis. Microbes within biofilms exhibit increased resistance to antimicrobial agents, and a significant amount of research has thus focused on gaining an understanding of how inter-domain interactions affect biofilm formation and the response to antimicrobial therapies. Candida albicans, a commensal and opportunistic pathogen of humans, is among the fungi most frequently identified in mixed-species biofilms. Here, we review interactions between C. albicans and bacterial species with which it is commonly isolated, namely Pseudomonas aeruginosa and Staphylococcus aureus in order to look into the spectrum of biologically relevant fungal–bacterial interactions that have been described.     

Probing of Microbial Biofilm Communities for Coadhesion Partners

Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method''s broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.     

生物被膜：益生菌肠道定植的新策略

益生菌可改善机体微生态平衡,在促进营养吸收、控制肠道感染和调节免疫功能等方面具有特殊的功效,但存在胃肠道环境难定植、口服生物利用度低等问题。生物被膜是多个细菌黏附于非生物或生物表面,分泌胞外聚合物(extracellular polymeric substances),并将自身包裹其中形成的一种有组织的细菌集团,包含胞外多糖(exopolysaccharides,EPS)、蛋白质、胞外DNA(extracellular deoxyribonucleic acid, eDNA)和脂质等多种组成成分,是一个具有三维立体空间结构的聚集体。被膜状态的益生菌较浮游菌在抗逆性、对抗病原菌和调节免疫功能等方面具有明显优势,这些特点为新型益生菌的开发提供了新的研究思路。本文阐述了被膜状态益生菌的优势,重点介绍了促进益生菌生物被膜形成的活性物及其形成机制,简述了益生菌生物被膜的安全性问题。当前,益生菌生物被膜的研究尚处于起步阶段,希望本文能为该领域未来的研究提供参考。     

In vitro anaerobic biofilms of human colonic microbiota

The human gastrointestinal tract hosts a complex community of microorganisms that grow as biofilms on the intestinal mucosa. These bacterial communities are not well characterized, although they are known to play an important role in human health. This study aimed to develop a model for culturing biofilms (surface-adherent communities) of intestinal microbiota. The model utilizes adherent mucosal bacteria recovered from colonic biopsies to create multi-species biofilms. Culture on selective media and confocal microscopy indicated the biofilms were composed of a diverse community of bacteria. Molecular analyses confirmed that several phyla were represented in the model, and demonstrated stability of the community over 96 h when cultured in the device. This model is novel in its use of a multi-species community of mucosal bacteria grown in a biofilm mode of growth.     

Biofilm development and enhanced stress resistance of a model,mixed-species community biofilm

Most studies of biofilm biology have taken a reductionist approach, where single-species biofilms have been extensively investigated. However, biofilms in nature mostly comprise multiple species, where interspecies interactions can shape the development, structure and function of these communities differently from biofilm populations. Hence, a reproducible mixed-species biofilm comprising Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was adapted to study how interspecies interactions affect biofilm development, structure and stress responses. Each species was fluorescently tagged to determine its abundance and spatial localization within the biofilm. The mixed-species biofilm exhibited distinct structures that were not observed in comparable single-species biofilms. In addition, development of the mixed-species biofilm was delayed 1–2 days compared with the single-species biofilms. Composition and spatial organization of the mixed-species biofilm also changed along the flow cell channel, where nutrient conditions and growth rate of each species could have a part in community assembly. Intriguingly, the mixed-species biofilm was more resistant to the antimicrobials sodium dodecyl sulfate and tobramycin than the single-species biofilms. Crucially, such community level resilience was found to be a protection offered by the resistant species to the whole community rather than selection for the resistant species. In contrast, community-level resilience was not observed for mixed-species planktonic cultures. These findings suggest that community-level interactions, such as sharing of public goods, are unique to the structured biofilm community, where the members are closely associated with each other.     

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.     

Applications of quorum sensing in biotechnology

Many unicellular microorganisms use small signaling molecules to determine their local concentration. The processes involved in the production and recognition of these signals are collectively known as quorum sensing (QS). This form of cell–cell communication is used by unicellular microorganisms to co-ordinate their activities, which allows them to function as multi-cellular systems. Recently, several groups have demonstrated artificial intra-species and inter-species communication through synthetic circuits which incorporate components of bacterial QS systems. Engineered QS-based circuits have a wide range of applications such as production of biochemicals, tissue engineering, and mixed-species fermentations. They are also highly useful in designing microbial biosensors to identify bacterial species present in the environment and within living organisms. In this review, we first provide an overview of bacterial QS systems and the mechanisms developed by bacteria and higher organisms to obstruct QS communications. Next, we describe the different ways in which researchers have designed QS-based circuits and their applications in biotechnology. Finally, disruption of quorum sensing is discussed as a viable strategy for preventing the formation of harmful biofilms in membrane bioreactors and marine transportation.     

High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions,Useful for Exploring Interspecific Bacterial Interactions

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Development of a multispecies oral bacterial community in a saliva-conditioned flow cell

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.     

Human oral cavity as a model for the study of genome-genome interactions

The enormous diversity of culturable bacteria within the oral microbial community coupled with experimental accessibility renders the human oral cavity a valuable model to investigate genome-genome interactions. The complex interactions of oral bacteria result in the formation of biofilms on the surfaces of the oral cavity. One mechanism thought to be important in biofilm formation is the coaggregation of bacterial partners. In this paper, we examine the role of coaggregation in oral biofilms and develop protocols to elucidate the spatial organization of bacterial species retained within oral biofilms. To explore these issues, we have employed two experimental systems: the saliva-coated flowcell and the retrievable enamel chip. From flowcell studies, we have determined that coaggregation can greatly influence the ability of an oral bacterial species to grow and be retained within the developing biofilm. To examine the spatial architecture of oral biofilms, fluorescent in situ hybridization protocols were developed that successfully target specific members of the oral microbial community. Together, these approaches provide insight into the development of oral biofilms and expand our understanding of genome-genome interactions.     

细菌生物膜研究技术

细菌生物膜是细菌生长过程中为适应生存环境而在固体表面上生长的一种与游走态细胞相对应的存在形式。只要条件允许,绝大多数细菌都可以形成生物膜。一旦形成了生物膜细菌就具有极强的耐药性,在医疗、食品、工业、军事等诸多领域给人类社会带来了严重的危害,造成巨大的经济损失。因此,细菌生物膜已成为全球关注的重大难题,也是目前科学界研究的前沿和热点。本文结合细菌生物膜研究技术的最新进展,重点介绍了几种常用生物膜发生装置及检测量化技术,并对其原理及优缺点进行了讨论。     

Cross- species communication in bacterial world

Biofilms are the compact association of micro organisms and the communication processes in these biofilms are always a wonder. Electrical and chemical signaling mechanism are the key to understand the bacterial communication network. Quorum sensing so far has been able to explain the coordinated motion of bacteria through its chemical signaling mechanism. Bacteria residing within biofilm communities are trivial to communicate. But the recent observation in 2017 by Humphries et al. has revealed that the ion channels enabled electrical signaling mechanism can be as powerful as to attract the distant cells i.e., this signaling mechanism are capable of holding a long range behavior. As a result long range cross species communication in the bacterial world have been possible. This substantial outcome has brought this field into a new paradigm to investigate the complex co-existence of biofilm communities and distant cells with a possible scope of application in synthetic biology. In this present article, we briefly describe this new signaling mechanism and how it gives rise to a long range communication ability in bacterial communities.     

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.     

Interspecies interactions within oral microbial communities.

While reductionism has greatly advanced microbiology in the past 400 years, assembly of smaller pieces just could not explain the whole! Modern microbiologists are learning "system thinking" and "holism." Such an approach is changing our understanding of microbial physiology and our ability to diagnose/treat microbial infections. This review uses oral microbial communities as a focal point to describe this new trend. With the common name "dental plaque," oral microbial communities are some of the most complex microbial floras in the human body, consisting of more than 700 different bacterial species. For a very long time, oral microbiologists endeavored to use reductionism to identify the key genes or key pathogens responsible for oral microbial pathogenesis. The limitations of reductionism forced scientists to begin adopting new strategies using emerging concepts such as interspecies interaction, microbial community, biofilms, polymicrobial disease, etc. These new research directions indicate that the whole is much more than the simple sum of its parts, since the interactions between different parts resulted in many new physiological functions which cannot be observed with individual components. This review describes some of these interesting interspecies-interaction scenarios.     

Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.     

Biofilms as a Mode of Existence of Bacteria in External Environment and Host Body: The Phenomenon,Genetic Control,and Regulation Systems of Development

Studies of the last decade have shown that most bacteria exist in natural ecosystems as specifically organized, attached to substrates biofilms rather than as freely floating plankton cells. The formation of these biofilms is a complex and highly regulated process. The development of biofilm communities is a primary strategy of bacterial survival not only in the external environment but also in the bodies of infected macroorganisms. In these organisms, bacteria are joined by complicated cell–cell associations, which makes them functionally similar to multicellular organisms. In the present review, we consider the structural organization of biofilms, factors affecting initiation of the biofilm formation, differential expression of bacterial genes at various stages of the biofilm development and their regulation. The significance of studies in this field for medicine, in particular, for prevention and protection against pathogenic bacteria, is discussed.