We determined the biofilm matrix types produced by S. saprophyticus recovered from infection and food-related setting based on their composition and explored its genetic basis in the population.

1. BACKGROUND
Biofilm formation is an important step in bacterial
colonization, adaptation and multiplication in a variety of
environments. It enhances pathogenicity and antibiotic
resistance in pathogens thereby contributing to disease
development in both animals and humans [1, 2]. S.
saprophyticus is a uropathogen that causes 10-20% of
uncomplicated urinary tract infection (UTI) worldwide. Its
ability to colonize and adhere to the bladder epithelium of
humans and immunocompromised individuals and the
capacity to form biofilm are important steps in the diseases
development [3, 4, 5]. However, the composition of S.
saprophyticus biofilms and its genetic basis remains unclear.
In this study, we determined the biofilm matrix types
produced by S. saprophyticus recovered from infection and
food-related setting based on their composition and explored
its genetic basis in the population.
• Bacterial strain collection: From a collection of 460 isolates, we selected 62,
representing all sub-clusters and belonging to two S. saprophyticus lineages;
the isolates were recovered from infection (n=41) and pig food-related
environment (n=21).
• Biofilm formation assay: All isolates were tested for ability to produce
biofilm using the modified polystyrene microtitre plates in static condition
[6]. Positive control: RP62A.
• Biofilm detachment assay: The preformed biofilm was treated with
Proteinase K (100 µg/ml) , DNAse I (100 µg/ml) and sodium periodate
(50mM). Each disruptor was added to the preformed biofilm which was
incubated for 3h at 37oC [7]. Absorbance (A595) was measured after biofilm
disruption. Strong or moderate biofilm producer which after disruption
became weak or none biofilm producer was deemed to be composed of the
component.
• Genomic sequencing and analysis. Draft genomes of the 63 S.
saprophyticus strains were obtained by Illumina MySeq. The genomes were
annotated using Prokka (v 1.13) [8] and pangenome was defined using Roary
(v 3.12) [9] with ≥85% blastp clustering cut-off with split-paralogues.
Genome wide association study (GWAS) was used to determine the
association between genomic, demographic and phenotypic data using
Scoary (v1.6.16) [10].
METHODS
RESULTS
CONCLUSIONS
•Biofilm formation in the S. saprophyticus population is an important phenotype for adaptation to their environments irrespective of the genetic background.
•There was a high variability in the composition of the biofilm formed by S. saprophyticus.
•All strains produced biofilms composed of protein, but the most common type contained protein, eDNA and polysaccharides.
•The biofilm components appear to differ between food-related and infection isolates, suggesting that modulation of biofilm composition could be a key step in S. saprophyticus virulence.
High variability in the composition of the biofilm formed by Staphylococcus saprophyticus from different origins
Opeyemi U. Lawal1,2, Marta Barata1, Ons Bouchami1,2, Peder Worning3, Mette D. Bartels3, Maria Luísa Goncalves4, Paulo Paixao5, Elsa F. Goncalves6, Cristina Toscano6,
Henrik Westh3, Maria João Fraqueza7, Hermínia de Lencastre2,8, and *Maria Miragaia1
1Laboratory of Bacterial Evolution and Molecular Epidemiology, Instituto de Tecnologia Química e Biológica (ITQB-NOVA) António Xavier, Oeiras, Portugal, Oeiras, Portugal. 2Laboratory of Molecular Genetics, ITQB-NOVA. 3Hvidovre University Hospital, Hvidovre, Denmark.
4SAMS Hospital, Lisbon, Portugal. 5Hospital da Luz, Lisboa, Portugal. 6Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal. 7CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal,
8The Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, United States.
*Presenting author: miragaia@itqb.unl.pt
Biofilm matrix composition in S. saprophyticus
Biofilm matrix phenotypes in S. saprophyticus were
associated to the source of isolates but not to
genetic background
1. Jeong, DW et al 2016. Int. J Food Microbiol. 236:9-163. 2. Speziale, P et
al. 2014 Front Cell Infect Microbiol 4:1-10; 3. Becker, K et al. 2014, Clin.
Microbiol. Rev. 27: 870-926; 4. Flores-Mireles, AL et al. 2015 Nat. Rev.
Microbiol 13:269-284; 5. Martins, KB et al. 2019 Front Microbiol 10:1-9. 6.
Vukovic et al. 2007, APMIS 891-999; 7. Fagerlund, A et al. 2016 Front
Microbiol 7:1-15; 8. Seeman, T 2014 Bioinformatics 30:2068-2069; 9. Page,
AJ et al. 2015 Bioinformatics 31: 3691-3693; 10. Brynildsrud, O et al. 2016
Genome Biol 17:1-9
REFERENCES
ACKNOWLEDGEMENTS
Biofilm matrix phenotypes in S. saprophyticus
population are multifactorial
Figure 1 - Matrix composition of the preformed biofilm of 62 S. saprophyticus strains recovered from human colonization and infection and food related
environment. (A) Remaining biofilm (%) after disruption. (B) Distribution of S. saprophyticus strains based on the biofilm composition.
Figure 2 – Distribution of biofilm matrix phenotypes among different
(A) genetic background and (B) sources of S. saprophyticus isolates.
34 38
7
10
12
14
46
38
0
20
40
60
80
100
Lineage G Lineage S
%
Isolates
A
• Two autolysins (Atl), fibronectin binding protein (Aas), lipase (Ssp), elastin binding
protein (EbpS) were present in all the isolates.
• Surface proteins were found in different proportions: SasF (85%), UafA (56%), SasD
(3%)
• Serine proteases were found in different proportions: SdrH (85%), SdrE (66%), SplE
(44%), SdrC (39%) and SraP (5%).
• icaC was ubiquitous; icaR was present only in isolates belonging to lineage G
(n=40).
• Complete ica gene cluster (icaADBCR) was present in three UTI isolates in the
collection while one isolate recovered from food carried icaADBC and no icaR.
• There was no direct association between the carriage of any particular gene and
the matrix type or the amount of biofilm produced.
Figure 3 - Maximum likelihood tree of 63 S. saprophyticus strains showing the
source, genetic lineages and distribution of genes putatively associated with
formation and matrix components of biofilm.
35
6
13
2
43
0
10
20
30
40
50
P
r
o
t
e
i
n
P
r
o
t
e
i
n
_
D
N
A
P
r
o
t
e
i
n
_
P
o
l
y
s
a
c
c
h
a
r
i
d
e
P
o
l
y
s
a
c
c
h
a
r
i
d
e
P
r
o
t
e
i
n
_
D
N
A
_
P
o
l
y
s
a
c
c
h
a
r
i
d
e
%
Isolates
Biofilm matrix composition
0
20
40
60
80
100
RP62A
KS71
KS79
KS88
KS96
KS98
KS103
KS104
KS105
KS11
KS110
KS114
KS115
KS121
KS123
KS125
KS127
KS128
KS133
KS14
KS147
KS180
KS208
KS220
KS227
KS229
KS233
KS234
KS242
KS248
KS25
KS264
KS268
KS284
KS290
KS292
KS295
KS297
KS298
KS36
KS300
KS301
KS305
KS310
KS312
KS313
KS314
KS319
KS328
KS332
KS338
KS342
KS351
KS352
KS356
KS357
KS358
KS365
KS371
KS373
KS384
KS387
KS410
KS430
%
Biofilm
remaining
after
disruption
Staphylococcus saprophyticus isolates
Human colonization and infection Food-related environment
A B
• Proteinase K disrupted the biofilm of more than 90% (n=57/62) of the strains and sodium periodate and DNAse disrupted 39% (n=24/62) and 34% (n=21/62),
respectively (see Fig. 1A).
• A decrease in the quantity of biofilm produced was found in >80% and 74% (n=31/41) of strains, even though they remained strong or moderate producer after
disruption with sodium periodate and DNAse I treatment, respectively.
• Overall, 43% (n=27/62) were composed of protein-eDNA-polysaccharide, 35% (n=22/62) were composed of only protein, 13% (n=8/62) were composed of protein-
polysaccharide and 6% (n=4/62) were composed of protein-eDNA (Figure 1B).
• There was diversity in biofilm phenotypes within S. saprophyticus
population.
• There was no significant difference between biofilm matrix phenotype
among S. saprophyticus genetic lineages.
• Biofilm matrix composed of only protein and protein-polysaccharide were
associated with isolates from infection (44%; n = 18/41, p = 0.0028, 17%; n
= 7/41, p = 0.03).
• Protein-eDNA-polysaccharides based biofilm were associated with isolates
from food-related environment (62%; n = 13/21, p = 0.0043) (Figure 2B).
44
19
5
10
17
10
34
62
0
20
40
60
80
100
Infection Food-related environment
%
Isolates
B
OUL was supported by Ph.D. grant PD/BD/113992/2015 from the Fundação
para a Ciência e Tecnologia (FCT). This work was partially supported by
project PTDC/FIS-NAN/0117/2014, project PTDC/CVT-CVT/29510/2017,
project PTDC/BIAMIC/ 31645/2017; Projects LISBOA-01-0145-FEDER-
007660 (Microbiologia Molecular, Estrutural e Celular) and
UID/Multi/04378/2019) funded by FEDER funds through COMPETE2020 -
Programa Operacional Competitividade e Internacionalização (POCI); by
ONEIDA project (LISBOA-01-0145-FEDER- 016417) co-funded by FEEI -
“Fundos Europeus Estruturais e de Investimento” from “Programa
Operacional Regional Lisboa2020” and by national funds through FCT;
Operacional Competitividade e Internacionalização, Programa Operacional
Regional de Lisboa (FEDER) and Fundação para a Ciência e a Tecnologia.
#163 – Session: II2. Mol. Microbiol & Microbial Physiol.