Table Of ContentSpringer Series on Biofilms
Karishma S. Kaushik
Sophie E. Darch Editors
Multispecies
Biofilms
Technologically Advanced Methods
to Study Microbial Communities
fi
Springer Series on Bio lms
Volume 12
SeriesEditors
Kendra P. Rumbaugh, Department of Surgery, Texas Tech University, Health
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TomCoenye,DepartmentofPharmaceuticalAnalysis,GhentUniversity,Ghent,
Belgium
Springer Series on Biofilms presents carefully refereed volumes on selected topics
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microbiology,biofilmsareofintenseinteresttomanyotherscientists,becausethey
affectwholeecosystems,andlieattherootofchronicbacterialinfections.
(cid:129)
Karishma S. Kaushik Sophie E. Darch
Editors
fi
Multispecies Bio lms
Technologically Advanced Methods to Study
Microbial Communities
Editors
KarishmaS.Kaushik SophieE.Darch
DepartmentofBiotechnology MorsaniCollegeofMedicine,Departmentof
SavitribaiPhulePuneUniversity MolecularMedicine
Pune,India UniversityofSouthFlorida
Tampa,FL,USA
ISSN1863-9607 ISSN1863-9615 (electronic)
SpringerSeriesonBiofilms
ISBN978-3-031-15348-8 ISBN978-3-031-15349-5 (eBook)
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Contents
3M’sofMulti-SpeciesBiofilms:MicrobialPathogens,
Microenvironments,andMinimalistLaboratoryApproaches
toStudyMulti-SpeciesBiofilmsUnderMicroenvironmental
Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
RadhikaDhekane,DeeptiBandaru,NijamuddinShaikh,
AnoushkaGholap,SnehalMurumkar,MeetaliBarhate,RutujaUgale,
UtkarshaTikhole,SnehalKadam,VandanaMadhusoodhanan,
andKarishmaS.Kaushik
MethodsfortheVisualizationofMultispeciesBiofilms. . . . . . . . . . . . . . 35
CéliaF.Rodrigues,JontanaAllkja,LuziaMendes,andAndreiaS.Azevedo
InVitro,InVivo,andExVivoModelstoStudyMixed
BacterialandFungalBiofilms. . . .. . . . . .. . . . .. . . . . .. . . . .. . . . . .. 79
AndreiaS.Azevedo,JontanaAlljka,SunilKumar,AwanishKumar,
SusanaBrás,andCéliaF.Rodrigues
CurrentandEmergingInVitroandInVivoBiofilmModels
inInvestigatingFungal-BacterialPolymicrobialCommunities. . . . . . . . 125
H.M.H.N.Bandara
InvolvementofBacterialOuterMembraneVesiclesinCell–Cell
InteractionsandTheirRoleinMulti-SpeciesCommunities. . . . . . . . . . 165
S.Mathur,H.Ortega,C.Pawlyshyn,andJ.W.Schertzer
PolymicrobialBiofilmModels:TheCaseofPeriodontalDisease
asanExample. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
KatherineAnsbro,CherFarrugia,GrahamP.Stafford,
andJoannaShepherd
v
vi Contents
PolymicrobialBiofilmsinCysticFibrosisLungInfections:
EffectsonAntimicrobialSusceptibility. . . . . . . . . . . . . . . . . . . . . . . . . . 231
AnnaClaraMilesiGaldino,MyleneVaillancourt,DianaCeledonio,
andPeterJorth
High-ResolutionMethodstoStudyMicrobialInteractions
inBiofilms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
KristenDominguez,AlexaGannon,CarolineD.Miller,
andSophieE.Darch
UnityinDiversity:InterkingdomPolymicrobialBiofilmsinDisease. . . . 309
SriramVarahan
’ fi
3M s of Multi-Species Bio lms: Microbial
Pathogens, Microenvironments,
and Minimalist Laboratory Approaches
fi
to Study Multi-Species Bio lms Under
Microenvironmental Conditions
RadhikaDhekane,DeeptiBandaru,NijamuddinShaikh,AnoushkaGholap,
SnehalMurumkar,MeetaliBarhate,RutujaUgale,UtkarshaTikhole,
SnehalKadam,VandanaMadhusoodhanan,andKarishmaS.Kaushik
1 Biofilm Infections
Microbial biofilms are associated with a range of clinical infection states, such as
cystic fibrosis, otitis media, periodontitis, and non-healing wounds. In contrast to
free-livingorplanktonicforms,biofilmsconsistofmicrobialaggregatessurrounded
by a self-produced extracellular matrix (O’Toole et al. 2000). In infection states,
biofilms are observed as three-dimensional, heterogenous bacterial communities
growing in close association with the infection microenvironment, which includes
host cells, matrix components, chemical and nutrient gradients, and biophysical
factors (Kadam et al. 2019). In addition to host tissue, bacterial biofilms also exist
onabioticsurfacessuchascatheters,dentalimplants,prostheticjoints,endotracheal
tubes, breast implants, and contact lenses (Raad and Hanna 2002; Imamura et al.
2008; Fernandes and Dias 2013; Abdallah et al. 2014). The process of biofilm
Radhika Dhekane, Deepti Bandaru, Nijamuddin Shaikh, Anoushka Gholap, Snehal Murumkar,
MeetaliBarhate,RutujaUgaleandUtkarshaTikholecontributedequallywithallother
contributors.
R.Dhekane·N.Shaikh·A.Gholap·S.Murumkar·M.Barhate·R.Ugale·U.Tikhole·
K.S.Kaushik(*)
DepartmentofBiotechnology,SavitribaiPhulePuneUniversity,Pune,India
e-mail:[email protected]
D.Bandaru
CentreforHealth,ResearchandEducation,Southampton,UK
S.Kadam
HullYorkMedicalSchool,UniversityofHull,Hull,UK
V.Madhusoodhanan
NCL-InnovationPark,Pune,India
©TheAuthor(s),underexclusivelicensetoSpringerNatureSwitzerlandAG2023 1
K.S.Kaushik,S.E.Darch(eds.),MultispeciesBiofilms,SpringerSeriesonBiofilms
12,https://doi.org/10.1007/978-3-031-15349-5_1
2 R.Dhekaneetal.
formation begins with the attachment of microbial cells or small aggregates of
microbes to a biotic or abiotic surface, or to each other as clumps of bacteria.
Following this initial attachment, microbes secrete extracellular polymeric sub-
stances(EPS),inwhichtheyproliferatetoformmicrocolonies(ahallmarkofbiofilm
formation).Mature biofilmsdisperse, intheformofsinglemicrobial cellsorsmall
aggregates, to seed new sites (Donlan 2001a). Importantly, microbes in biofilms
display several features distinct from their planktonic counterparts, notably,
increased tolerance to immune clearance and antibiotic treatments (Hoiby et al.
2010;Gunnetal.2016;YanandBassler2019).
2 Multi-Species Biofilm Infections
Clinical biofilm infections are often associated with multi-species biofilm commu-
nities,whichcanincludearangeofbacterialspecies,aswellasbacterialandfungal
species (Elias and Banin 2012). Infact, certain studies report that up to 60–70% of
microbialinfectionsareassociatedwithmixed-speciesbiofilms(Tytgatetal.2019;
Rao et al. 2020). In chronic wound biofilms, Pseudomonas aeruginosa and Staph-
ylococcus aureus are the most common co-pathogens, often found along with
commensal and anaerobic bacteria such as Corynebacterium spp. and
Propionibacterium spp. (Wolcott et al. 2016). In lung biofilms associated with the
genetic disorder Cystic Fibrosis (CF), bacterial communities consist of
P. aeruginosa, S. aureus, Burkholderia spp., Streptococcus spp., Prevotella, and
Fusobacterium,andtheirrelativeabundanceisknowntovarybetweenindividuals
(Jean-Pierreetal.2021).S.aureuscolonizestheCFlungatanearlyage,followedby
colonization of P. aeruginosa in later years. The colonization by S. aureus in turn
paves way for secondary colonization by P. aeruginosa, with the latter eventually
outcompeting S. aureus. This pre-colonization is correlated with worsened health
outcomes for patients with chronic lung infections (Alves et al. 2018). Similarly,
biofilmsassociatedwithoralcavityconditionssuchasdentalcariesandperiodontal
diseases are well-known to possess a diverse microbiota, often composed of
100–300 different species (Griffen et al. 2012). In the context of oral biofilms,
inter-kingdom interactions between bacteria and fungi are of notable significance.
For example, Candida albicans, an opportunistic yeast, provides a scaffold for
S. aureus establishment on abiotic surfaces, otherwise known to poorly form
biofilmsonsuchsurfaces(Petersetal.2012).TheC.albicansscaffoldinitshyphal
form is seen to supplement nutrients and enhance the vancomycin resistance of
S. aureus in a multi-species setting, underscoring the cooperative nature of the
interaction (Harriott and Noverr 2010). In another example of oral multi-species
biofilms, Streptococcus gordonii, a commensal and key bacterial species in dental
plaque,increasesthehyphaldevelopmentofC.albicans,interactingwiththefungal
elements via specific proteins, and thereby establishing a bacterial-fungal inter-
kingdom multi-species biofilm (Harriott and Noverr 2011). Taken together, multi-
species biofilm infections are characterized by the presence of multiple microbial
3M’sofMulti-SpeciesBiofilms:MicrobialPathogens,Microenvironments,... 3
species,incloseassociationwitheachotherandthehostmicroenvironment(Fig.1).
Further,interactionsacrossthemulti-speciesbiofilmstatecanaffecttheprogression,
recalcitrance,virulence,andtherapeuticoutcomeoftheinfection.Thisunderscores
the importance of studying not only the diversity of pathogens in these infections,
butalsothefeaturesoftheinfectionstateunderhost-relevantconditions.
3 Microbial Pathogens in Multi-Species Biofilms
In the following section, we present an overview of select multi-species biofilm
infections,withafocusonthediversityofmicrobialpathogensthatcouldcontribute
to the infection state, and interspecies interactions. While not all inclusive, this
segmentprovidesadescriptionofthemostcommonmulti-speciesbiofilminfections
andtheirmostwell-studiedfeatures.
3.1 Chronic Wound Biofilms
It is well established that chronic wounds harbor a plethora of microbes that
contribute directly and indirectly to their non-healing state. The protein-rich sub-
strate and supply of nutrients in the wound bed serve as an ideal environment for
biofilmformation(Malicetal.2009).Microbialcommunitiesinwoundsoftenexist
as multi-species biofilms, comprising both Gram positive and negative species,
including Staphylococcus aureus, Pseudomonas aeruginosa, β-hemolytic Strepto-
cocci, Enterococcus spp., Klebsiella pneumoniae, Acinetobacter baumannii,
Enterobacter spp. (ESKAPE pathogens), coagulase-negative Staphylococci and
Proteusspecies(Bowleretal.2001).Inadditiontodispersedaggregatesbelonging
todifferentbacterialspecies,clinicalbiopsiesofchronicwoundshavealsorevealed
well-mixed bacterial communities, including the presence of dual-species within a
singlebiofilmaggregate(Jamesetal.2008;Maloneetal.2017;Johanietal.2017;
Suryalethaetal.2018).Alongwithaerobes,anaerobicspeciesarebelievedtoforma
significantproportionofthewoundmicrobialburden.However,owingtochallenges
withisolationandidentification,theycontinuetoremainpoorlystudied.Fungialso
comprise a part of the wound consortium, including Candida, Curvularia, and
Malassezzia spp. (Kalan and Grice 2018). Interactions between different bacterial
species,betweenbacterialandfungalspecies,andmicrobialcomponentsandmicro-
environmental factors are known to affect the progression and outcome of chronic
woundinfections.
