A requirement for flow to enable the development of Ureaplasma parvum biofilms in vitro

To use a flow‐based method to establish, quantify and visualize biofilms of Ureaplasma parvum.

S e e h t t p://o r c a .cf. a c. u k/ p olici e s. h t ml fo r u s a g e p olici e s.Co py ri g h t a n d m o r al ri g h t s fo r p u blic a tio n s m a d e a v ail a bl e in ORCA a r e r e t ai n e d by t h e c o py ri g h t h ol d e r s .

Introduction
Mollicutes include several human and veterinary relevant pathogens.These organisms differ from many bacteria by lacking a cell wall, being pleomorphic in shape, harbouring a genome with reduced coding capacity and an essential requirement for sterols.The bacteria within the genus Ureaplasma are unique among Mollicutes due to their sole utilization of urea in the acquisition of energy (Robertson et al. 2002).The conversion of urea to ammonia and carbon dioxide results in an increase in the surrounding pH, which comes at the cost of viability when grown in vitro and therefore giving a limited window in which it is possible to work with viable organisms (Masover et al. 1977).Two human associated Ureaplasma species exist, Ureaplasma parvum and Ureaplasma urealyticum.Among adults, ureaplasmas colonize mucosal membranes within the genital tract of around 40-80% of women and 30% of men (Waites et al. 2005).Although colonization among asymptomatic individuals is common, several disease states have been associated.Ureaplasmas can ascend the female genital tract during pregnancy, leading to in utero infection, chorioamnionitis and subsequent premature birth (Sweeney et al. 2017;Pavlidis et al. 2020).Infections of the genital tract among men that result in non-gonococcal urethritis (NGU) is still under debate, with evidence of species of ureaplasma present, host adaptive immune response and bacterial load being key factors in disease outcome (Beeton et al. 2019).Infections with Ureaplasma sp. of the genital tract can be persistent even in the presence of antimicrobial therapy suggesting a potential means to evade antimicrobials and the immune clearance (Khosropour et al. 2015).
Capacity to form a biofilm, quantification of biomass and visualization of these three-dimensional structures have been described for a number of Mollicutes such as Mycoplasma pneumoniae, Mycoplasma pulmonis and Mycoplasma genitalium (Simmons et al. 2007(Simmons et al. , 2013;;Simmons et al. 2007Simmons et al. , 2013;;Daubenspeck et al. 2020;Daubenspeck et al. 2020).To date, there is very little information regarding biofilm formation by the urea utilizing mycoplasmas in the genus Ureaplasma (Garc ıa-Castillo et al. 2008;Pandelidis et al. 2013) with all published methods focusing on static models with a lack in quantification or visualization of the biofilms formed.
Here, we discuss the essential requirement for flow for U. parvum to develop a biofilm.Using this system, it was possible to give the first quantification of biofilm biomass produced by U. parvum utilizing a flow-based system.Furthermore, we present confocal scanning laser microscopy and scanning electron micrograph images of U. parvum which have been grown using this method.

Strains and media used
All experiments were conducted with U. parvum strain HPA5.Cultures were grown in Ureaplasma Selective Media (Mycoplasma Experience, Surrey, UK) within microtitre plates covered with an adhesive sealing film to prevent leaching of ammonia to adjacent wells.To ensure inoculation with viable, actively growing U. parvum, 10-fold dilutions were grown overnight.The next morning the lowest dilution in which a colour change had occurred was used for inoculation into the flow cell.

Growth curve experiment
A 10-ml culture of U. parvum HPA5 was established with viable cells from an overnight dilution.Cultures were incubated under static conditions at 37°C for 30 h.Every three hours the titre of U. parvum was quantified by performing serial 10-fold titration in USM within microtitre plates which were sealed and incubated for 48 h until colour change had ceased (Beeton et al. 2012).The lowest dilution in which a colour change occurred was designated as 1 CCU.Therefore, by calculating back through the dilution series, it was possible to extrapolate the number of CCU in the original culture.In addition to CCU quantification, absorbance readings at 550 nm, as surrogate marker for growth due to media alkalisation and detectable equivalence point of phenol red indicator present in the growth media, were determined by spectroscopy (Tecan Infinite M200) (Heath et al. 2020).

Set up and running of the DTU flow cell
The DTU flow cell was prepared as described elsewhere (Crusz et al. 2012;Tolker-Nielsen and Sternberg 2014).In brief, 24 9 55-mm borosilicate glass D 263 TM coverslips (VWR, Lutterworth, UK) were adhered to the DTU flow cell with transparent silicone.The flow cell was connected to a Masterflex LS peristaltic pump and the system was flooded with 1% v/v hypochlorite for 4 h.The system was purged three times with air and then flushed with sterile water for 16 h, prior to displacing the sterile water with USM.Inlet tubes were surface sterilized with an alcohol wipe, and a 27G insulin needle (VWR) was used to inoculate each chamber with 150 µl of viable overnight culture (resulting puncture holes were sealed with silicone).For direct comparison of static and flow conditions, two channels were inoculated with the same overnight preparation.Both flow cells were left static for four hours to allow for attachment of U. parvum.Following the attachment phase, only one channel was subjected to flow rate of 0Á01 ml min À1 , while the media in the second channel remained static.Duration of the experiment allowed to progress for a further 24 h, all media and equipment being contained incubated at 37°C.

Confocal scanning-laser microscopy imaging of biofilms and quantification of bound cells
At 28-h post-inoculation, 200 µl of sterile phosphatebuffered saline (PBS) was gently flushed through the channels to remove any non-adherent cells.Two hundred microliter of 5 mmol l À1 Syto9 in PBS (ThermoFisher, UK) was then injected into each channel.Samples were incubated in the dark at 37°C for 30 min followed by rinsing with a further 200 µl of PBS to remove any background Syto9.Biofilms were the visualized on a Nikkon Eclipse 80i microscope using 485-nm excitation and 498nm emission filters.Five random sections were selected for each chamber, and Z-stack images were taken.
Following imaging, channels were triturated vigorously with 200 µl of USM to dislodge bound cells.Bacterial viability was quantified by determining the CCUs by titration as outlined above.

Preparation of biofilms for visualization by scanning electron microscopy
Flow cell experiments were repeated exactly as outlined above, except that post-removal of non-adherent cells at 28-h post-inoculation, coverslips were removed from the chamber and fixed with 2Á5% glutaraldehyde in 0Á1-mol l À1 sodium cacodylate buffer.Samples were postfixed with 1% osmium tetroxide, dehydrated in an acetone series (50-100%) and chemically dried in hexamethyldisilazane.Samples were sputter coated with a thin layer of chromium and imaged using a field emission scanning electron microscope (JEOL JSM6301F).

Statistical analysis
Biomass was determined by COMSTAT for analysis (Heydorn et al. 2000).Statistical analysis by t test was undertaken in GraphPad Prism ver.5.01.

U. parvum rapidly lose viability following log phase under static conditions
Ureaplasma parvum was grown under static conditions for 30 h with measurements of viability and media colour change as a marker for media alkalisation taken every 3 h (Fig. 1a).Culture media remained orange for 9 h with a mean absorbance value of 1Á7; however, evidence of alkalization was apparent by increased absorbance by the phenol red at 12 h (light red in colour) and the media appearing transparent and cerise red in colour after 15 h as indicated by an absorbance reading of 2Á8 (Fig. 1b).In parallel, viability assessment (quantified by titration in USM) showed a lag phase between 0 and 3 h followed by an exponential increase in number between 3 and 12 h.No stationary phase was observed, showing the susceptibility of U. parvum to toxic by-products as viability rapidly declined between 15 and 24 h.

Evaluation of U. parvum biofilms under flow and static conditions
Using the DTU flow cell system U. parvum HPA5 was inoculated into two channels.Following adhesion, the first channel was subject to flow of 0Á01 ml min À1 , while the second channel was left under sterile static conditions.Sporadic clusters of biofilm were observed throughout the channel subjected to flow conditions (Fig. 2a,b).Comet-like tail structures were observed from the colonies, usually observed tapering with the direction of flow.No biofilm was observed within the channel under static conditions.Figure 2c shows the evaluation of Z-stack images with COMSTAT that confirmed a statistically significant level of biomass under flow conditions (0Á599 µm 3 /µm 2 AE 0Á152) compared to growth under no flow (0Á008 µm 3 /µm 2 AE 0Á010).The number of viable cells attached within the channel after 28-h postinoculation was also determined by CCU quantification (Fig. 2d).Under flow conditions 4 9 10 8 CCU per ml were recovered as compared to no detectable viable growth when subjected to static conditions.

Scanning electron microscopy of U. parvum biofilms grown under flow conditions
Scanning electron microscopy was used to obtain detailed high-magnification images of U. parvum HPA5 cells grown under flow conditions for 28 h (Fig. 3).Cells attached to the glass cover slip were sporadic after preparation for imaging (Fig. 3a).Cells which remained were associated with an extracellular matrix linking cells together (Fig. 3b).At a magnification of 15 0009 (Fig. 3c) cells were pleomorphic in shape and variable in size.On closer inspection, some cells also displayed signs of budding with the possibility of membrane vesicles formation and conjugative apparatus-like appearing protrusions (Fig. 3d; indicated with * and ^, respectively).

Discussion
Ureaplasma sp. are unique in their essential requirement for urea in the production of ATP (Robertson et al. 2002).Although human-associated ureaplasmas are recognized causes in a number of persistent infections such as those associated with preterm birth, neonatal lung disease and suggested links with NGU among men, very little is known regarding the ability to form biofilms.The first indications to suggest Ureaplasma sp. have the capacity to form a biofilm was presented by Garc ıa-Castillo et al. (2008) and then subsequently by Pandelidis et al. (2013).Both studies utilized a static peg-lid-based approach to grow biofilms of clinical isolates of Ureaplasma sp.whereby lids were transferred to fresh media following the change of culture media from orange to red.Using this methodology, the studies found 82 and 95% of clinical isolates had the capacity to form biofilms, respectively.Although most isolates were found to form biofilms using this method, in some instances, some isolates were unable to.It is not clear if this was either true absence of biofilm-forming capacity, or an artefact of overincubation and viability loss between transfer to fresh media resulting in biofilm failure (Masover et al. 1977).Inferring from our viability data with U. parvum HPA5, it suggests that the viability window from the point of detectable colour change is very narrow: bacterial viability peaks before full visible colour change to red has occurred (Fig. 2b).Therefore, media change 6-9 h after this point would see significant loss of viable cells and strains with shorter generation time would fair even worse.
Due to the limitations of a static system resulting from toxic metabolite accumulation and subsequent drop in cell viability, we reasoned that a flow-based system would sustain the development of Ureaplasma biofilms.By using the DTU flow cell method, it was possible to run parallel culture, with the same inoculum, under static and flow conditions.Confocal scanning laser microscopy was used to obtain the first images of biofilms formed by U. parvum in our axenic culture.Biofilms took on a sporadic microcolony appearance and did not form large areas of biomass unlike that seen with other bacteria (Crusz et al. 2012).It is not known if the development of these microcolonies are the result of cell aggregates settling and continuing to expand through cell division as observed with M. pneumoniae tower formation (Feng et al. 2020).This socially distanced phenotype of sporadic microcolonies could be an adaptive trait in order to prevent the accumulation of localized toxic metabolites by large areas of biomass upon a surface.As ureaplasmas exist as obligate parasites (requiring nucleotides, sterols and other key nutrients they cannot synthesize), this would also prevent damage to the cells on which they are bound.In addition to this sporadic microcolony phenotype, a comet-like tail appearance was apparent which has described in other bacteria under flow, such as Hemophilus influenzae (Cho et al. 2015).This finding was in stark contrast to the channel which was subjected to static conditions which lacked any detectable biofilm.This absence of biofilm was not surprising based on the growth curve viability data.
A common method for quantifying biofilm utilizes crystal violet staining of biomass, which has been successfully used with other mycoplasmas (Simmons and Dybvig 2015).Our previous attempts to utilize this method suggested that it was not sensitive enough due to the low levels of biomass (data not shown).We therefore utilized image data Z-stacks obtained from CLSM using COM-STAT analysis, to give the first quantifiable measurement of biomass formed by U. parvum.A second level of quantification was the recovery of high titre of viable organisms when subjected to flow, past the point at which viability was lost within the static channel.
Using SEM, it was possible to get the first highresolution images of ureaplasma biofilms when grown in axenic culture.Previously Torres-Morquecho et al.
(2010), used SEM to visulise ureaplasmas attached to A549 lung epithelial cells.The void in SEM images of ureaplasma attached to surfaces in axenic culture may be related to the difficulties of balancing high cell titres to localized toxic concentrations of ammonia and CO 2 production unless flow or chelation can remove these byproducts.A further observation from the SEM images was the presence of budding and potentially the formation of membrane vesicles.Budding has previously been observed among ureaplasmas, but little is known with regards to the formation of membrane vesicles (Whitescarver and Furness 1975).Finally, from the SEM images, structures which resembled suspected conjugation pili were apparent.Literature on horizontal gene transfer among ureaplasmas is limited; however, attempts to use qPCR target panels to serotype clinical ureaplasmas established that extensive horizontal gene transfer rendered this impossible (Xiao et al. 2011), which was then confirmed by detailed whole genome analysis of 19 strains of ureaplasma subsequently (Paralanov et al. 2012).Identification of a mobile antibiotic resistance gene, tet(M) element, which is present on a transposable element has also been identified in both U. parvum and U. urealyticum (D egrange et al. 2008;Beeton and Spiller 2017).Although HPA5 is tet(M)-negative, the presence of these pili may suggest the presence of other uncharacterized transposable elements as suggested by the presence of the UU372 DDE-type transposase (NP_078206.1), the integrase-recombinase protein UU404 (NP_078239.1) and the tyrosine recombinase XerC (UU222; NP_078055.1)found within the whole genome sequence of HPA5 (unpublished data).Although data suggest the presence of transposable elements within the HPA5 genome, it should be emphasized that the presence of a conjugation pili is speculative and based on observations from the electron micrograph.Further work is underway to examine if this is a true finding or an artefact from the preparation of the samples.
In this study, we present a flow-based method for growing biofilms of U. parvum superior to static methods, given the removal of toxic-metabolite mediated cell death.Using this flow-based approach, it was possible to use fluorescent and electron microscopy to gain the high-resolution images of the resulting biofilm.Confocal laser scanning microscopy images were then used to give the first quantification of biomass formed by U. parvum.With this platform, it is now feasible to interrogate the role of individual surface associated adhesins, such as the multiple banded antigens, for their role in biofilm formation, as well as the capacity to investigate biofilm-mediated resistance to antimicrobials.
Pl e a s e n o t e: C h a n g e s m a d e a s a r e s ul t of p u blis hi n g p r o c e s s e s s u c h a s c o py-e di ti n g, fo r m a t ti n g a n d p a g e n u m b e r s m a y n o t b e r efl e c t e d in t his ve r sio n.Fo r t h e d efi nitiv e ve r sio n of t hi s p u blic a tio n, pl e a s e r ef e r t o t h e p u blis h e d s o u r c e.You a r e a d vis e d t o c o n s ul t t h e p u blis h e r's v e r sio n if yo u wi s h t o cit e t hi s p a p er. Thi s v e r sio n is b ei n g m a d e a v ail a bl e in a c c o r d a n c e wit h p u blis h e r p olici e s.

Figure 1
Figure 1 The relationship between Ureaplasmaparvum HPA5 viability and change in the colour of culture media over 30 h under static conditions.(a) Ureaplasma parvum culture at inoculation represented by orange media (left) compared with positive culture (red) with no visible sign of turbidity and unknown cell titre or viability (right).(b) The change in media colour over time quantified by absorbance readings at 550 nm (red line) and viability of cells quantified by determining the CCU (black line) every 3 h.CCU = colour changing units.

Figure 2
Figure 2 Presence of Ureaplasma parvum HPA5 biofilms formed under flow conditions.Ureaplasma parvum biofilms were grown under flow (0Á01 ml min À1 ) and no flow (0 ml min À1 ) conditions for 28 h.(a & b) Representative Syto9 stained biofilms grown under flow conditions with direction of flow from right to left.(c) Z-stacks were taken and compared with no flow conditions with biomass quantified with COMSTAT2 image analysis software.(d) The number of viable cells associated with the surface was quantified by flushing PBS through the chamber following visualisation and subsequently determining the number of colour changing units (CCU).

Figure 3
Figure 3 Scanning electron micrographs of Ureaplasma parvum HPA5 biofilms formed under flow conditions.Ureaplasma parvum biofilms were grown under flow conditions for 28 h and prepared for SEM (a) with indication of an extracellular matrix (b).Cells were highly pleomorphic exhibiting budding (c).White arrows indicated by * suggest the presence of membrane vesicles and white arrows indicated by a ^indicate the possibility of conjugation pilus (d).