Community context influences the conjugation efficiency of Escherichia coli

Abstract In urinary tract infections (UTIs), different bacteria can live in a polymicrobial community consisting of different species. It is unknown how community members affect the conjugation efficiency of uropathogenic Escherichia coli. We investigated the influence of individual species often coisolated from urinary infections (UTI) on the conjugation efficiency of E. coli isolates in artificial urine medium. Pairwise conjugation rate experiments were conducted between a donor E. coli strain containing the pOXA-48 plasmid and six uropathogenic E. coli isolates, in the presence and absence of five different species commonly coisolated in polymicrobial UTIs to elucidate their effect on the conjugation efficiency of E. coli. We found that the basal conjugation rates of pOXA-48, in the absence of other species, are dependent on the bacterial host genetic background. Additionally, we found that bacterial interactions have an overall positive effect on the conjugation rate of pOXA-48. Particularly, Gram-positive enterococcal species were found to enhance the conjugation rates towards uropathogenic E. coli isolates. We hypothesize that the nature of the coculture and physical interactions are important for these increased conjugation rates in an artificial urine medium environment.


Introduction
Antimicr obial r esistance (AMR) poses a significant challenge to global public health (Murray et al. 2022 ).The intense use of antibiotics has led to the emergence and spread of multidrug resistance in pathogenic bacteria (Polianciuc et al. 2020 ).This threatens the effectiveness of antibiotics, and therefore our ability to cure infections (Prestinaci et al. 2015, Aslam et al. 2018 ).
Bacteria can acquire AMR by horizontal exchange of genetic material among related or unrelated bacterial species, in a process r eferr ed to as 'horizontal gene tr ansfer' or HGT (Oc hman et al. 2000, Hall et al. 2017 ).The exchange of genetic material between microbes can occur in various wa ys , often by a process called conjugation (Furuya and Lowy 2006 ).Conjugation involves the physical contact between donor and recipient cells and typically a self-transmissible or mobilizable plasmid (Ochman et al. 2000 ).
Conjugative or mobilizable plasmids are the most common tr ansmission v ectors for AMR genes (Boerlin and Reid-Smith 2008, P artridge et al. 2018, Ar es-Arr o y o et al. 2022 ) and the major drivers of HGT within bacterial communities (Bottery 2022 ).AMR genes and HGT have been observed within the human microbiome.A major hotspot for antibiotic resistance is, for instance, the gut microbiome of humans and animals (San Millan 2018 ), where rich dynamics of plasmid transfer have been observed (Frazão et al. 2023 ).
Although the ecology and functioning of microbial communities ar e typicall y studied in one specific environment at a time (Smillie et al. 2011 ), it is known that the rate of HGT is str ongl y dependent on the abiotic and the biotic environments (Sessitsch et al. 2023 ).For instance , resource a v ailability and temper atur e (P allar es- Vega et al. 2021 ) or other abiotic factors such as salt stress (Beuls et al. 2012 ), can significantly affect the rate at which HGT via conjugation occurs.Biotic factors, such as the presence of ecological interaction partners, can affect the spread of conjugative plasmids within and between host species (Bottery 2022 ).Additionally, the horizontal transmission of plasmids can be limited by bacterial diversity due to the 'dilution effect'; i.e. the phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species (Kottara et al. 2021 ).The microbial context, such as the presence of competitors, can also determine the cost and benefits of conjugative plasmid maintenance (Sünderhauf et al. 2023 ).Mor eov er, ecological inter actions can alter factors suc h as gr owth r ate and population densities, whic h together can affect the cost of plasmid carriage and conjugation rates (Duxbury et al. 2021 ).
It is still an open question to what extent bacterial interactions affect the transfer of antibiotic resistance by HGT via conjugation in bacterial comm unities.Giv en that complex communities are difficult to study, we investigate this question for a simple and tr actable, yet r ele v ant, system consisting of bacterial species often coisolated from elderly patients diagnosed with urinary tract infections (UTIs) (Croxall et al. 2011b ).The prevalence of AMR in suc h comm unities is high (Croxall et al. 2011b ), and there has been an increase in AMR and multidrug resistance in recent years (Trautner et al. 2022 ).
In such communities, Gram-positive species live together with Gr am-negativ e species (de Vos et al. 2017, Zandbergen et al. 2021 ), but the importance of Gr am-positiv e species for UTIs is often ov erlooked.Yet, Gr am-positiv e bacteria ar e an important cause of nosocomial infections (Furuno et al. 2005, Cong et al. 2019 ).Enterococci, for instance , ha ve been shown to facilitate polymicrobial infections, leading to more complicated pathogenesis and poor er pr ognoses (Chong et al. 2017, Barshes et al. 2022 ), and they can compromise the efficacy of antimicrobial agents by promoting colonization, pr olifer ation, and persistence of diverse pathogenic bacteria (Xu et al. 2023 ).Furthermore, they can act as reservoirs for the transmission of AMR and virulence determinants (Coburn et al. 2007, Xu et al. 2021 ).
Here , we in vestigate the effect of ecological interactions between Esc heric hia coli and other bacterial species often identified in pol ymicr obial UTIs on the conjugation rate of pO XA-48 to w ar ds uropathogenic E. coli .pOXA-48 is a plasmid with a broad host r ange, carrying the r esistance gene bla OXA-48 that confers resistance to multiple β-lactam antibiotics (Poirel et al. 2004(Poirel et al. , 2012 ) ), including carba penems, whic h ar e last-r esort antibiotics used to tr eat m ultidrug-r esistant infections (Br adley et al. 1999, P a pp-Wallace et al. 2011 ).It is an important conjugative plasmid in the clinical setting, known for its r a pid dissemination within hospital patients and has a worldwide distribution (Pitout et al. 2019, León-Sampedro et al. 2021 ).
Specifically, we study the effect of ecological interactions on the conjugation efficiency in uropathogenic E. coli isolates, by performing pairwise conjugation assays in the presence of Enterococcus faecium , Enterococcus faecalis , Staphylococcus simulans , Pseudomonas aeruginosa , and Proteus mirabilis in artificial urine medium (AUM) .All isolates, except for P. aeruginosa , were collected from elderly patients who were diagnosed with polymicrobial UTIs (Croxall et al. 2011b ).It is important to mention that the term conjugation 'efficiency' is used as a qualitative description of the ability of recipient E. coli isolates to take up pOXA-48 plasmid.On the other hand, conjugation 'rate' is used to refer to the quantitative assessment of the transfer of pOXA-48 plasmid to uropathogenic E. coli .

Bacterial isolates
Nine E. coli isolates were selected from a previous study where samples were collected from elderly patients diagnosed with pol ymicr obial UTIs (Cr o xall et al. 2011b ).The y were selected based on their sensitivity to ampicillin.Multi locus sequence typing, follo w ed b y phylotyping (Beghain et al. 2018 ) of these uropathogenic E. coli isolates was performed pr e viousl y by Croxall et al. ( 2011a ).Initial conjugation experiments aimed at testing their ability to take up pOXA-48 plasmid resulted in six final uropathogenic E. coli isolates that were used as recipients in pairwise mating assays in the presence of UTI community members.Plasmid transfer in these isolates was confirmed by Pol ymer ase Chain Reaction 'PCR' with specific primers for pOXA-48 plasmid (see the section 'DNA extraction and PCR in Materials and methods').
Four community members were collected from the same study as the uropathogenic E. coli isolates (Croxall et al. 2011b ), and were also selected upon their sensitivity to ampicillin.These belonged to thr ee Gr am-positiv e species: E. f aecium , E. f aecalis , and S. simulans , and one Gr am-negativ e species: P. mirabilis .Additionall y, we inv estigate the inter action with the Gr am-negativ e P. aeruginosa , because this species is fr equentl y r ecov er ed fr om pol ymicr obial UTIs (de Vos et al. 2017 ).Because the pol ymicr obial UTI P. aeruginosa isolates in our collection were highly resistant to ampicillin, we used PAO1, which was less resistant to ampicillin.

Efficiency of uropathogenic E. coli to take up pOXA-48 plasmid via conjugation in LB
This protocol was used to assess, rather qualitatively, the ability of the uropathogenic E. coli isolates to take up the pOXA-48 plasmid, and it was adapted from Alonso-del Valle et al. ( 2021Valle et al. ( , 2023 ) ). Donor β3914 and recipient E. coli str ains wer e str eaked on CHRO-Magar plates supplemented with kanamycin 30 μg/ml (Sigma) and 300 μM DAP (Sigma), for the donor; and no antibiotic for the r ecipients, giv en that they were sensitive to most antibiotics used for the treatment of UTIs .T he plates were incubated overnight at 37 • C. The next day, three independent colonies were picked from each isolate and grown overnight in 2 ml of Lysogeny broth (LB) at 37 • C and continuous shaking at 200 r pm.Donor cultur es wer e grown with 30 μg/ml kanamycin and 300 μM DAP.
The following day, the ov ernight cultur es, whic h wer e at that time in stationary phase, were mixed in a 5:1 donor-to-recipient volume ratio.This relationship was established after testing sever al donor-to-r ecipient pr oportions, and this r atio r esulted as the most effective for plasmid transfer.The experiment was performed in triplicates; 50 μl of the donor culture and 10 μl of the r ecipient cultur e wer e gentl y mixed by pipetting in 0.5 ml tubes.The full 60 μl droplets were spotted in the middle of LB agar (Sigma) plates without antibiotics but with 300 μM DAP (Sigma), and left to air-dry in the flow cabin, after which they were incubated at 37 • C for 4 h to r ecov er tr ansconjugants .T his mating time is short enough to reduce the chances of secondary conjugation e v ents fr om tr ansconjugants to r ecipients and the impact of potential differences in donor, recipient, and transconjugant growth rates on conjugation frequency determination (Alonso-del Valle et al. 2023 ).The controls consisted of 60 μl of isolated cultures of donor or r ecipient; eac h in triplicates, whic h wer e also spotted in the middle of individual LB agar (Sigma) plates with 300 μM DAP (Sigma).
After incubation, a metal loop was used to scoop out the biomass of each of the droplets, which were immediately washed and resuspended by pipetting in tubes containing 2 ml of sterile 0.9% NaCl solution.These were further diluted in serial 10-fold dilutions from 10 1 until 10 7 using a 96-well plate: 200 μl of resuspension was added to the first well and the remaining wells contained 180 μl of 0.9% NaCl solution.Then, 20 μl from the first well was taken and mixed with the next well.This step was repeated for all columns of the well plate.A m ultic hannel pipette was used to transfer 10 μl from each dilution at the first quarter of a round a gar plate, whic h was tilted 90 • to let the eight dr oplets slide down until the end of the plate.Every donor and recipient mix, as well as the controls (donors in isolation and recipients in isolation), were plated on tr ansconjugant-selectiv e LB a gar plates with ampicillin 100 μg/ml wher e onl y tr ansconjugants should gr ow.As an additional negativ e contr ol, e v ery donor and recipient mix, as well as the controls (donors in isolation and recipients in isolation) were plated on LB agar plates with kanamycin 30 μg/ml (Sigma) without DAP, to make sure that neither donor nor recipients in isolation nor transconjugants would grow.
After ov ernight incubation, gl ycer ol stoc ks wer e made fr om the tr ansconjugants.Giv en that this was a r elativ el y crude, qualitative method to assess conjugation, a more quantifiable method was later applied to determine pOXA-48 conjugation rates in the presence and absence of UTI community members in AUM.

DN A extr action and PCR
To confirm plasmid transfer to the six E. coli isolates, DN A w as extracted using a previously in-house developed ultra-fast DNA extraction method for E.coli described in Brons et al. ( 2020 ).Primers for amplifying the resistance gene bla OXA-48 for β-lactam antibiotics on the pOXA-48 plasmid were adopted from Poirel et al. ( 2004 ).A 20-mer forw ar d primer, designated Oxa-48 Fw (5 -TTG GTG GCA TCG ATT ATC GG-3 ) was combined with a 21-mer reverse primer, designated Oxa-48 Rev (5 -GA G CA C TTC TTT TGT GAT GGC-3 ).This primer combination was tested and optimized.
PCR mixtur es wer e pr epar ed with the following components: 5.0 μl of 10x Roche buffer (Roche, Basel, Switzerland), 0.8 μl of 50 mM MgCl 2 (Merc k, Darmstadt, German y), 1.0 μl of 100% dimethyl sulfoxide, 0.5 μl of 20 mg/ml bovine serum albumin (Merck), 1.0 μl of 10 mM deo xyribon ucleoside triphosphate mix, 1.0 μl of 10 μM of each primer, and 0.2 μl of 5 U/ μl Taq DNA Polymer ase (Roc he).Molecular biology-gr ade water (Thermo Fisher Scientific, Waltham, USA) was added to a total volume of 50 μl in a 0.2-ml micr ofuge tube.Finall y, 1.0 μl of template DN A w as added.The mixtures were incubated in a Master c ycler Nexus PCR thermal cycler (Eppendorf, Hambur g, German y) with the following pr ogr am: initial denatur ation of double-str anded DNA for 5 min at 95 • C; 35 cycles consisting of 1 min at 95 • C, 30 s at 56 • C, and 2 min at 72 • C; and extension for 7 min at 72 • C.
All amplification products were analyzed by electrophoresis in 1.0% (w/v) a gar ose gels, follo w ed b y ethidium bromide staining (1.2 mg/l ethidium bromide in 1 × Tris-acetate-EDTA) (Sambrook et al. 1989, Mullis 1990 ), destaining (1 × Tris-acetate), and visualization under UV.Amplicons of 743 bp in size were detected, and no side products were observed, confirming plasmid transfer to the six E. coli isolates.to six uropathogenic E. coli isolates in the presence and absence of five members of the pol ymicr obial UTI comm unity in AUM media.Each conjugation experiment was performed in a single day and consisted of six assays: the 'basal conjugation rate' assay with only donor and recipient; and the pairwise 'community member assays' wher e eac h of the fiv e other species wer e ad ded indi vidually to the donor and recipient combination.Every assay was performed with three biological replicates.

Conjuga tion r a tes of pOXA-48 to uropa thogenic E. coli with and without community members on AUM
A scoop from −80 • C glycerol stocks was taken to grow overnight cultures of donor strain β3914, recipient E. coli and community members with 2 ml of 1x AUM.The donor strain was grown with 30 μg/ml kanamycin (Sigma) and 300 μM DAP (Sigma).The recipient and community member strain cultures had no ad diti ves.They were incubated for 24 h at 37 • C with continuous shaking at 200 rpm.
After 24 h, optical density measurements at 600 nm (OD600) measur ements wer e taken of all strains using a 1-ml culture in a disposable cuvette in a spectrophotometer.The population sizes wer e inferr ed fr om these OD600 measur ements, by diluting the cultures below 0.4 OD and calculating their true OD based on the dilution factor used for each strain.Each culture was then further diluted to obtain a starting population size that, in combination, would maintain a 5:1:1 OD600 ratio between donor, recipient, and comm unity member, r espectiv el y.These pr oportions wer e the same as used for the 'Efficiency of uropathogenic E. coli to take up pOXA-48 plasmid via conjugation in LB' protocol (see the section 'Materials and methods').Specifically, the OD600 values used were 1, 0.2, and 0.2, r espectiv el y; except in the case of E. faecium and E. f aecalis , wher e the OD was always lower than 0.2.
For the assessment of the basal conjugation rates of pOXA-48 plasmid to E. coli , 50 μl of donor and 50 μl of r ecipient wer e added and gently mixed in a 0.5-ml tube, preserving a 5:1 OD600 ratio.This combination was the control of the experiment, the basal conjugation rate.For the assessment of the effect of other species fr equentl y co-occurring in pol ymicr obial UTIs on the conjugation rates, 50 μl of donor and recipient were also added to a 0.5-ml tube with an additional volume from the individual community member culture that was dependent on the OD, but always close to 50 μl, preserving the 5:1:1 OD600 ratio of donor, recipient, and community member.If the OD of the undiluted culture was < 0.2, whic h was al ways the case with E. faecium and E. faecalis , then exactly 50 μl of it was added to the tube .T her efor e, the total dr oplet volume for the basal conjugation assay was 100 and 150 μl for the individual community member assa ys .For mixing, vortexing w as av oided, and the tubes w er e gentl y struc k se v er al times.All dr oplets wer e spotted in the middle of 1x AUM agar plates containing DAP.These plates wer e pr epar ed using 50% Micro agar (15 g/l) (Duchefa Biochemie), 50% 2x AUM and 300 μM DAP.The dr oplets wer e left to dry and incubated to allow for conjugation at 37 • C for 1 h.This was performed in triplicates for e v ery combination of donor + recipient and of donor + recipient + single other species.
After 1 h of incubation, the plates were removed from the incubator.A sterile toothpick was used to cut out the piece of agar with the dr oplet.Subsequentl y, the a gar segment was crushed and resuspended in 1 ml sterile 0.9% NaCl solution.Each tube was inverted and gently shaken 30 times to wash off the cells fr om the a gar.Further 10-fold dilutions until 10 4 wer e made befor e plating 100 μl of the resuspensions to obtain countable colonies.Transconjugant colonies were obtained either at undiluted or 10 1 diluted r esuspensions.Contr ol, as well as e v ery combination of donor + recipient + single other species were plated in two types of selective plates: LB agar (Sigma) with 30 μg/ml kanamycin and DAP and CHROMagar (Condalab) with 100 μg/ml ampicillin; which were used to obtain CFU/ml counts of β3914 donor strain, and to distinguish E. coli transconjugants from any other species able to grow in ampicillin; and on the nonselective plates made of CHRO-Magar (without DAP) (Condalab) to count E. coli recipient isolates.They were left overnight at 37 • C and colonies were counted the next day.
The pOXA-48 plasmid conjugation rate was estimated using the formula: T/ ( D • R • t ) (Lopatkin et al. 2016, Huisman et al. 2022 ) where the CFU/ml of the transconjugants is r epr esented by T ; D are CFU/ml the donor, and R are CFU/ml of the recipient.The time in which conjugation took place is r epr esented by t , and it w as alw ays 1 h; the a ppr oximate time needed for pOXA-48 to produce transconjugants (León-Sampedro et al. 2021 ), while k ee ping on-plate growth to a minimum.

Conjuga tion r a tes of pOXA-48 to uropa thogenic E. coli in artificial urine spent media
To assess the indir ect inter action effect of the enterococci isolates, via metabolic compounds in their exudates (de Vos et al. 2017 ), on the conjugation efficiency of E. coli , conjugation experiments between donor strain β3914 and two E. coli isolates; B and F (Fig. 1 ), were performed on conditioned media agar plates containing spent medium from two enterococcal isolates, and the contr ols on AUM a gar plates .T he two E. coli isolates wer e c hosen based on contrasting conjugation rates (Fig. 1 ).The protocol follo w ed the same methodology as in the pr e vious section; namel y, mixing together 50 μl of each culture at an OD600 ratio of 5:1, plating the full droplet in the middle of the plates and incubating at 37 • C for 1 h; with the main difference being the plate composition.
Specifically, spent media were recovered from two UTI isolates; E. faecalis and E. faecium by inoculating bacterial glycerol stocks in 200 ml 1x AUM in Erlenmeyer flasks shaking at 200 rpm at 37 • C for 48 h.Afterw ar ds, cultures w ere distributed into 50 ml culture tubes and centrifuged for 15 min at 4800 × g at room temperature .T he resulting supernatants were filtered twice with bottle filter tops; 0.45 μm and 0.2 μm filters, r espectiv el y.To make sure that all bacteria were filtered out, spent medium was plated on CHROMagar plates, and incubated at 37 • C for 24 h, whereafter the plates sho w ed no bacterial gro wth.
The conditioned media agar plates were prepared using 50% Micr o a gar (15 g/l) (Duc hefa Bioc hemie), 25% spent media, and 25% 3x AUM (including 1x concentration AUM salts).Control plates did not contain spent media in and consisted of 50% Micro agar (15 g/l), 25% 1x AUM, and 25% 3x AUM (including 1x concentration AUM salts).The end concentration of AUM in the conditioned media plates depended on how m uc h nutrients were depleted by the bacteria, between 0,75x AUM (if all nutrients were consumed) and 1x AUM depending (if no nutrients were consumed).All plates contained 300 μM DAP to ensure the survival of donor bacteria.

Sta tistical anal ysis
Statistical anal yses wer e performed in R v4.1.2(R Cor e Team 2021 ) and the pac ka ge DescTools v0.99.50 (Signor ell 2024 ).We used log 10 -transformed conjugation rates for analyses after visually confirming normality and homoscedasticity of the transformed data ( Supplementary Figs 2 and 3 ).We tested the differences in basal conjugation rates of pOXA-48 plasmid to different E. coli recipient strains with a one-way ANOVA ( P < .05).To assess the impact of community members (Conjugation rates of pOXA-48 to uropathogenic E. coli with and without community members on AUM) and exudates of community members (Conjugation rates of pOXA-48 to uropathogenic E. coli in artificial urine spent media) on conjugation rates, we first tested whether the effect of E. coli r ecipient str ain and comm unity member wer e independent (tw o-w ay ANOVA, P < .05).Because we found a significant interaction between the two main effects, we proceeded to assess the impact of community members on conjugation rates associated with each recipient strain separately (one-way ANOVA followed by Dunnett's test for comparison to control, P < .05).

Results
To investigate the impact of other community members often coisolated from polymicrobial UTIs on the conjugation efficiency of uropathogenic E. coli , we compared the pOXA-48 reception rate through conjugation of isolated uropathogenic E. coli with that of uropathogenic E. coli in the presence of other species commonly isolated from polymicrobial UTIs.

Efficiency of uropathogenic E. coli to take up pOXA-48 plasmid via conjugation in LB
Specifically, we performed conjugation experiments between E. coli donor strain β3914 and nine uropathogenic E. coli isolates, using plasmid pOXA-48; initially on LB media (see the section 'Materials and methods').Three out of these nine E. coli isolates didn't take up the plasmid.Of the six uropathogenic E. coli isolates that did take up the plasmid (Fig. 1 A-F, see the section 'Materials and methods'), they did so with different conjugation efficiencies; indicated by the qualitative assessment of the maximum dilution that obtained transconjugants ( Supplementary Table 1 ).

Conjuga tion r a tes of pOXA-48 to uropa thogenic E. coli with and without community members on AUM
Of the six uropathogenic E. coli isolates that could take up the plasmid, we tested the differential effects on the conjugation rates with donor strain β3914 in the absence and in the presence of eac h of fiv e other species fr equentl y coisolated in pol ymicr obial UTIs in AUM: E. faecium , E. faecalis , S. simulans , P. aeruginosa , and P. mirabilis .Conjugation rate experiments were performed on AUM agar by bringing donor, recipient, and one other isolate of the abo ve-mentioned species , together in a droplet of conjugation mix, as described by León-Sampedro et al. ( 2021 We first analyzed the two main effects ( E. coli isolates and individual community members) across the whole experiment.We found that the effect of community members differs significantly for different E. coli isolates (tw o-w ay ANOVA, Df = 25, F value = 14.15, inter action: P < 2e-16).Mor eov er, we found that the basal conjugation rates, in the absence of community members, differ significantly between the six E. coli isolates (ANOVA, Df = 5, F value = 15.82,P = 6.4e-05).The conjugation rates of pOXA-48 from donor to recipient in isolation range within two orders of magnitude between the different isolates; from 4.7 × 10 −14 for E. coli isolate A, to 4.6 × 10 −12 for E. coli isolate F (Fig. 1 ).Assessing the effect of the bacterial interactions on the conjugation rate, we did not find that any of the other tested isolates inhibit the growth of either donor or recipient to such an extent that the variation in donors and recipients alone cannot explain the increase in conjugation rate ( Supplementary Table 2 ).Only for E. coli isolates B and F , the changes of donors and recipients in the presence of E. faecium can partially explain the statistically significant changes in conjugation rate (Fig. 1 , one-way ANOVA and Dunnett's post hoc test, P < .05).
Rather, we found that bacterial inter actions gener all y hav e a positive effect on the conjugation rates.Particularly the Grampositiv e species E. f aecium and E. f aecalis , but also S. simulans contribute to this effect.For five of the six E. coli isolates, at least one Enterococcus species has a significant positive effect on the conjugation r ate, wher eas S. simulans has a positiv e effect on the conjugation rates in three of the E. coli isolates .T he Gram-negative species P. aeruginosa and P. mirabilis gener all y hav e a less prominent effect.P. mirabilis alters the conjugation rates in three of the six E. coli species, whereas P. aeruginosa only affects the conjugation rates in one E. coli isolate (Fig. 1 ; Supplementary Table 2 ).
The extent to which pairwise interactions changed the conjugation rates varied substantially between isolates.For instance, for E. coli isolate A, three UTI community members increased the conjugation rate by three orders of magnitude.For isolate C, the presence of both Enterococcus species increased the conjugation rate b y tw o or ders of magnitude.Yet, for E. coli isolate E we could not detect any significant changes to the conjugation rate due to the influence of any other of the UTI community members (Fig. 1 ).The magnitude of the variability of the conjugation rates was constant throughout the tested conditions ( Supplementary Fig. 1 ).
We find that the conjugation rate levels for the tested isolates do not correspond to their assigned phylogroups (Croxall et al. 2011a ) ( Supplementary Table 3 ).For instance, isolates A and D are both part of ECOR group B2 (Ochman and Selander 1984 ) as well as two of the isolates that did not take up the plasmid (based on the efficiencies of uropathogenic E. coli to take up pOXA-48 plasmid via conjugation in LB).One of the latter two belongs to the sequence type 131, a drug-r esistant ur opathogenic str ain of E. coli widely disseminated among both community and hospital patients (Lau et al. 2008 ).This suggests that specific genetic components other than phylogroup classification are important for the ability to take up the pOXA-48 plasmid.

Conjuga tion r a tes of pOXA-48 to uropa thogenic E. coli in artificial urine spent media
To investigate if the increased conjugation r ates, particularl y due to the presence of enterococci, were due to their exudates (for instance metabolic products produced), we tested whether the presence of spent media of E. faecium and E. faecalis can r eca pitulate the findings.Conjugation experiments were performed with two Figure 2. Relativ e differ ences in conjugation r ates of pOXA-48 plasmid into two E. coli recipient strains (B and F) in the absence (Control) and presence of spent media containing exudates from another UTI community member ( E. faecium and E. faecalis) .Small dots indicate indi vidual re plicate measur ements, lar ge dots r eplicate means.One r eplicate measur ement ( E. coli F with E. faecalis ) w as belo w the detection limit and was set to 1e-14 for the anal ysis.Comm unity members with a significant impact on conjugation rates (solid circles) relative to the control conjugation rate (dotted line) are indicated by solid circles (ANOVA, Dunnett's test, P < .05).
E. coli isolates (isolates B and F) on spent medium agar plates (see the section 'Materials and methods'), with the spent media from the two enterococci.These isolates were chosen based on the contrasting conjugation rates from Fig. 1 .
We found that the spent media experiment gener all y shows a rather small negative effect on the conjugation rate.Namely, in three combinations of E. coli under the influence of enterococci: E. coli B with E. faecalis and E. faecium , and E. coli F with E. faecium the effect was not significant and in one; E. coli F in the spent medium of E. faecalis , the effect was significant but negative .T herefore , this experiment cannot explain the marked positive effect on the conjugation rate in the presence of E. faecium and E. faecalis (Fig. 2 ; Supplementary Table 4 ) compared to the positive effects found in the AUM experiments.We have also tested these results under additional r efer ence conditions aimed at assessing the boundary conditions of nutrient depletion in the conditioned medium.Supplementary Fig. 4 shows that under these conditions, the relative effect of enterococci on the conjugation rates of pOXA-48 plasmid into two E. coli isolates B and F, was significant and negativ e in thr ee combinations: E. coli B with E. faecalis and E. coli F with E. faecalis and E. faecium , while for E. coli B with E. faecium the effect was not significant.This indicates that primary or secondary metabolites released by E. faecium and E. faecalis are unlikely to be the leading cause of the increased conjugation r ates observ ed in the coculture experiments.
It should be noted that Gr am-positiv e enter ococci hardl y gr ow in AUM (10 3 -10 4 CFU/ml), these r elativ el y low counts mimic their growth in urine (Flores-Mireles et al. 2015 ).These cells are therefor e pr esent in low numbers in the cocultur e conjugation r ate experiments on the AUM agar plates.Spent medium agar plates containing exudates from these cells ther efor e likel y contain a higher concentration of these metabolic exudates compared to the concentration of these exudates in the drops in the coculture conjugation experiments.We cannot rule out that these different concentrations in the different experiments may influence the effect on conjugation rates .T hat we do observe a marked increase in conjugation rates in the physical presence of enterococci, but not in the presence of their exudates, suggests that Gram-positive bacteria affect the conjugative transfer of AMR in uropathogenic E. coli , in a manner that is lik ely de pendent on the physical interaction of E. coli and the Gr am-positiv e species.

Discussion
This work is aimed at investigating the impact of species often coisolated from polymicrobial UTIs on the conjugation efficiency of uropathogenic E. coli.We show that these species can positiv el y affect the transfer of pOXA-48 plasmid from E. coli to uropathogenic E. coli in the low-nutrient AUM after 1 h of conjugation.Gr am-positiv e species, particularl y enter ococci, and also sometimes Gr am-negativ e species, suc h as P .aeruginosa and P .mirabilis increase the conjugation rates of pO XA-48 betw een E. coli .
Nine uropathogenic E. coli isolates wer e initiall y selected for conjugation with pOXA-48.Only six of these E. coli isolates took up the plasmid, as verified by PCR with pOXA-48 specific primers (see the section 'Materials and methods').The fact that each of them was associated with unique basal conjugation rates indicates that ther e ar e host-dependent genetic bac kgr ound inter actions that determine these r ates, whic h is in accordance with other findings (Alonso-del Valle et al. 2023 , Benz andHall 2023 ).Regarding the different sequence types and phylogroups assigned to the tested E. coli isolates, we find that the le v els of conjugation r ates, and e v en the ability to take up pOXA-48 plasmid, were not consistent with those .T hese r esults contr ast pr e vious liter atur e showing that specific phylogroups are associated with specific transferability of plasmids (Carattoli 2011 ), that are associated with particular pathogenic phenotypes or multidrug resistance, as is the case with sequence type 131 (Lau et al. 2008 ).
In general, the presence of a plasmid in a new bacterial host might come with fitness costs, for which single compensatory mutations are often sufficient to completely ameliorate such costs, suggesting that these are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expr ession le v el (Hall et al. 2021 ).For instance, plasmid-encoded extended-spectrum beta-lactamase gene acquisition in diverse E. coli lineages can drive strain-specific inter actions, suc h as c hr omosomal m utations affecting metabolic and regulatory functions that ultimately might affect plasmid stability and conjugation efficiency (Carrilero et al. 2023 ).In the wider context of bacterial communities, both the cost of plasmid carriage and its long-term maintenance in a focal strain are found to depend on the presence of competitors, and these interactions are species-specific too (Sünderhauf et al. 2023 ).
The conjugation rate experiments were performed in the lownutrient AUM to r eca pitulate an envir onment that is closer to the in vivo environment of the uropathogens .Otherwise , similar conjugation experiments are often performed in LB (Alonso-del Valle et al. 2021, León-Sampedro et al. 2021, DelaFuente et al. 2022 ) or viande-le vur e (Card et al. 2017, Duxbury et al. 2021 ), which ar e r ather ric h media and yield mostl y higher conjugation r ates.The conjugation rates we find are similar to conjugation rates of pOXA-48 in one strain of E. coli, cultured under anaerobic conditions and in rather poor M9 minimal media (León-Sampedro et al. 2021 ).We, ther efor e hypothesize that the similarly low basal conjugation rates result from the low nutrient en vironment.T his hypothesis was confirmed by testing the ability to take up pOXA-48 plasmid through conjugation via the rather crude qualitative dr oplet-dr oplet method in a rich LB medium (see the section 'Materials and methods'; Supplementary Table 1 ).
Conditioned medium experiments indicate that metabolic compounds in the exudates of the cocultured enterococcal species ar e unlikel y to be the leading cause of the increased transfer.T hus , E. coli growth and survival mediated by such exudates of enter ococci ar e also unlikely to be involved (Keogh et al. 2016 ).Mor eov er, to limit such potential growth effects we performed the incubation step of the conjugation rate experiments for only 1 h, whereas other studies often use longer incubation times (Alonso-del Valle et al. 2023 ).And lastly, because the conjugation rate is calculated based on the number of donors, recipients and transconjugants at the end of the 1-h conjugation incubation time (after the cogrowth of the donor β3914, recipient E. coli and community member on the agar plate), we conclude that growth r ate differ ences ar e not the cause of the marked increase in conjugation rate in the presence of these species.
Pr oximity between cells, suc h as that found in biofilms, is known to facilitate the spread of antibiotic resistance by promoting HGT (Fux et al. 2005 ) e.g.via quorum sensing, which leads to changes in gene expression, potentially expediting the acquisition of antibiotic resistance (Schroeder et al. 2017, Lin et al. 2021 ).Howe v er, if the increased conjugation rates would be caused by quorum sensing molecules released by enterococci, then we expected to observe an increase in conjugation rates in the presence of conditioned medium pr epar ed fr om enter ococci, and that was not the case.Our findings ther efor e suggest that the nature of the coculture and its direct interactions are important for these increased conjugation rates in AUM.Specifically, we hypothesize that physical contact or proximity (Stalder and Top 2016 ), between these species may play a role.It is worth mentioning that pOXA-48 is an Inc L/M plasmid type that encodes short, rigid pili known to have higher tr ansfer fr equencies on solid surfaces compar ed to liquid (Bradley 1980 ). Various types of cell-to-cell contact have been shown to be involved in promoting the transfer of genetic material within species (Morawska and Kuipers 2022 ).One alternative hypothesis would be that the presence of some Gr am-positiv e species is strengthening the interaction between the two E. coli strains (donor and recipient) as a sort of defense mechanism, limiting the dir ect inter action of the Gr am-positiv e 'intruder' with the interacting E. coli strains .T his is reminiscent of biofilm formation as a defense mechanism (Donlan andCosterton 2002 , Kumar et al. 2017 ).
The Gr am-negativ e species P. mirabilis , also had a positive effect on the conjugation efficiency for three of the E. coli isolates.
It is known that P. mirabilis raises the pH of the AUM medium (Broomfield et al. 2009, Chen et al. 2012, de Vos et al. 2017 ), and pH has been shown to influence conjugation rates (Alderliesten et al. 2020 ).Additionally, it may be that this raised pH leads to a stress r esponse; str ess r esponses ar e speculated to affect the conjugation rates (Johnsen and Kroer 2007 ).Additionally, we speculate that a potential explanation for E. coli str ain-specific r esponses in conjugation efficiency in the presence of UTI community members is related to the fact that some of the E. coli str ains hav e differ ent colon y mor phologies (mor e or less fuzzy or r ounded), whic h potentiall y r elates with these isolates being more or less 'sticky' than others .T hese ma y, ther efor e, be mor e pr one to pr oximate inter actions, whic h ar e r elated with incr eased uptake of plasmids (Robledo et al. 2022 ).For future studies, it would be of great interest to understand how multispecies interactions would affect conjugation efficiencies in E. coli .
Although successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission (Duxbury et al. 2021 ), the fact that uropathogenic E. coli are conjugatable at these le v els in an AUM environment suggests that the urinary tract and its urobiome, is a potential location where HGT takes place (Wolfe and Brubaker 2019, Jones et al. 2021, Kuznetsova et al. 2022, Montelongo Hernandez et al. 2022 ).
Finally, our findings on the increased conjugation rates of pOXA-48 to uropathogenic E. coli in the pr esence of, particularl y, Gr am-positiv e species underscor e that ecological inter actions ar e r ele v ant for the conjugative transfer of AMR, also in a urine-like environment.

Ac kno wledgements
We kindly thank Asheigh Griffin (University of Oxford) for donating P. aeruginosa PAO1.We thank Alv ar o San Millan (Centro Nacional de Biotecnología-Madrid) for providing the donor strain, plasmid, and protocol, as well as for discussions and for reading the initial version manuscript.We thank Alan McNally for sharing uropathogenic isolates and information on the phylotype of these E. coli isolates.We thank the Center for Information Technology of the University of Groningen for their support and for providing access to the Hábrók high performance computing cluster.We thank two anon ymous r e vie wers for their constructive comments on the manuscript.

Figure 1 .
Figure 1.Conjugation rates of pOXA-48 plasmid to six uropathogenic E. coli recipient strains (A-F) in the absence (control) and presence of one additional UTI community member ( E. faecium , E. faecalis , S. simulans , P. aeruginosa , and P. mirabilis ).Small dots indicate individual replicate measurements ( n = 3), large dots replicate means.Community members with a significant impact on conjugation rates relative to the basal conjugation rate (control, dotted line) are indicated by solid circles (ANOVA, Dunnett's test, P < .05).