Detection and identification of unknown streptococcal populations in clinical samples

Figures & data

Table I. Bacterial strains used in the study.

Species	Strain
Arthrobacter sp.	UdG001
Bacillus cereus	UdG_002
Bacillus megaterium	UdG_003
Bacillus subtilis	UdG_004
Campylobacter jejuni	ATCC 33291
Citrobacter sp.	CECT401
Enterobacter aerogenes	CECT684
Enterococcus avium	CECT968
Enterococcus columbae	CECT4798
Enterococcus durans	CECT411
Enterococcus faecalis	DMSZ20478
Enterococcus faecium	CECT410
Enterococcus munditii	CECT972
Enterococcus gallinarum	CECT970
Amylovora sppilophylus	UdG_005
Erythrobacter sp.	UdG_006
Escherichia coli	ATCC 10536
Lactobacillus sp.	UdG_007
Listeria innocua	CECT5376
Methylophilus methylotrophus	UdG_008
Micrococcus luteus	UdG_009
Mycobacterium avium	UdG010
Proteus mirabilis	UdG_170
Pseudomonas fluorescent	UdG011
Salmonella LT2	UdGLT2
Serratia marcescens	UdG 013
Shigella sonnei	CECT457
Staphylococcus aureus	ATCC 25923
Staphylococcus epidermidis	UdG_014
Streptococcus agalactiae	CECT183
Streptococcus anginosus	CECT948
Streptococcus bovis	DMSZ20480
Streptococcus mutans	CECT479
Streptococcus pyogenes	CECT598
Streptococcus sanguinis	CECT480
Streptococcus equinus	CECT213
Streptococcus salivarius	CECT805
Streptococcus oralis	CECT907
Streptococcus suis	CECT958
Streptococcus thermophilus	CECT986
Streptococcus uberis	CECT994
Streptococcus intermedius	CECT803
Streptococcus pneumoniae	CECT993
Streptococcus sobrinus	CECT4034
Streptococcus equi subsp. equi	CECT989
Streptococcus mitis	DMSZ12643

DMSZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; CECT, Colección Española de Cultivos Tipo; UdG, University of Girona collection.

Table II. Primers used in this study.

Primer	Sequence (5’–3’)	Target	Reference
27F	AGAGTTTGATCCTGGCTCAG	Eubacteria	Wang et al. (22)
357F	CCCTACGGGAGGCAGCAG	Eubacteria	Muyzer et al. (15)
1492R	ACGGTTACCTTGTTACGACTT	Eubacteria	Hayashi et al. (23)
623F	GTGGCGAAAGCGGCTCTCT	Streptococcus sp.	This work
623R	AGAGAGCCGCTTTCGCCAC	Streptococcus sp.	This work
1043F	CACTCTAGCGAGACTGCCG	Streptococcus sp.	This work
1043R	CGGCAGTCTCGCTAGAGTG	Streptococcus sp.	This work

F, forward; R, reverse.

Table III. Specificity test assay for primers 1043F-1492R on bacterial species from the genusStreptococcus and other phylogenetic groups.

Streptococcus species	PCR	Actinobacteria and Firmicutes	PCR	Proteobacteria	PCR
Streptococcus agalactiae	+	Arthrobacter spp.	−	Campylobacter jejuni	−
Streptococcus sanguinis	+	Bacillus cereus	−	Citrobacter spp.	−
Streptococcus anginosus	+	Bacillus megaterium	−	Escheriehia coli	−
Streptococcus mutans	+	Bacillus subtilis	−	Enterobacter aerogenes	−
Streptococcus pyogenes	+	Enterococcus avium	−	Amylovora sppilophylus	−
Streptococcus bovis	+	Enterococcus columbae	−	Erythrobacter spp.	−
Streptococcus equi subsp. equi	+	Enterococcus durans	–	Methylophylus metylophilus	–
Streptococcus sobrinus	+	Enterococcus faecium	−	Proteus mirabilis	−
Streptococcus equinus	+	Enterococcus gallinarum	−	Pseudomonas fluorescens	−
Streptococcus salivarius	+	Enterococcus munditii	−	Salmonella LT2	−
Streptococcus oralis	+	Lactobacillus spp.	−	Serratia marcescens	−
Streptococcus suis	+	Listeria innocua	−	Shigella sonnei	−
Streptococcus thermophilus	+	Micrococcus luteus	−
Streptococcus uberis	+	Mycobacterium avium	−
Streptococcus intermedius	+	Staphylococcus aureus	−
Streptococcus pneumoniae	+	Staphylococcus epidermidis	−
Streptococcus mitis	+	Enterococcus faecalis	+

Figure 1. DGGE profiles of 16S rRNA gene fragments from Streptococcus spp. from colon biopsies (A) and bronchoalveolar lavage samples (B). White dots indicate bands further subjected to re-amplification and sequencing. The percentage of urea + formamide solution is indicated (far right)

Table IV. Sequence identification of DGGE bands in GenBank and RDP II.

DGGE Band	Accession number	Size (pb)	Similarity (%)	Nearest sequence (NCBI)	Description	Source	Nearest strain (RDP-II)	S_ab Score	GenBank sequence
C13.a	EU563992	378	91	AF432136	Streptococcus oral clone	Tongue dorsum scrapings	Streptococcus mitis (T); ATCC 49456	0.919	AF003929
CRC16.C	EU563987	374	93	DQ346424	Uncultured Streptococcus	Human oral cavity	Streptococcus thermophilus (T); D SM 20617	0.842	X68418
CRC4.a	EU563979	276	94	DQ819119	Uncultured bacterium clone aab26a11	Digestive tract of zebrafish	Streptococcus thoraltensis (T) S6 9	0.934	Y09007
CRC16.a	EU563985	375	95	EF400504	Uncultured bacterium clone SJTU_E_14_21	Human fecal sample	Streptococcus pneumoniae (T); ATCC 33400	0.802	AF003930
CRC21.C	EU563999	379	95	EF404797	Uncultured bacterium clone SJTU_C_10_72	Human fecal sample	No significant match	< 0.700
CRC21.e	EU564001	377	95	DQ905141	Uncultured bacterium clone 002C-d6	Human fecal sample	No significant match	< 0.700
CRC16.b	EU563986	377	96	AM157451	Streptococcus salivarius	Human breast milk	Streptococcus thermophilus (T); D SM 20617	0.870	X68418
C19b	EU563995	338	96	EF071403	Uncultured Firmicutes clone M0027_118	Human colonic mucosa	Ehterococcus faecium (T); DSM20477	0.879	AJ276355
CRC21.a	EU563997	377	96	AY854277	Uncultured bacterium clone CATTLE_15	Herbivore gastrointestinaltra	No significant match	< 0.700
ClO.a	EU563984	373	97	DQ905402	Uncultured bacterium clone 013C-C10	Human fecal sample	Streptococcus thermophilus (T); D SM 20617	0.945	X68418
C19.a	EU563994	336	97	DQ677551	Uncultured Streptococcus sp. clone D01	Human saliva	Streptococcus mitis (T); ATCC 49456	0.960	AF003929
CRC21.d	EU564000	337	97	EF405369	Uncultured bacterium clone SJTU_G_02_85	Human fecal sample	No significant match	< 0.700
CRC21.f	EU564002	376	97	DQ905380	Uncultured bacterium clone 013C-A11	Human fecal sample	No significant match	< 0.700
CRC4.b	EU563980	283	97	EF404405	Uncultured bacterium clone SJTU_C_09_54	Human fecal sample	Streptococcus thermophilus (T); D SM 20617	0.935	X68418
CRC4.d	EU563983	376	97	DQ905141	Uncultured bacterium clone 002C-d6	Human fecal sample	No significant match	< 0.700
BL55.a	EU563978	358	97	AY959207.1	Uncultured bacterium clone rRNA434	Human vaginal epithelium	Enterococcus avium	0.902	AJ301825
BL59.b	EU563975	359	97	AY803232.1	Uncultured bacterium clone D112	Rhizoplane of Lemnaminor	No significant match	< 0.85
CRC16.e	EU563989	374	98	AY959090	Uncultured bacterium clone rRNA317	Human vaginal epithelium	Streptococcus intermedius (T); ATCC27335	0.968	AF104671
CRC16.g	EU563991	375	98	AY959090	Uncultured bacterium clone rRNA317	Human vaginal epithelium	Streptococcus intermedius (T); ATCC27335	0.954	AF104671
CRC21.g	EU564003	378	98	EF406903	Uncultured bacterium clone DSS_7DAYS_ C05_T7P	Mouse colon	No si gni ficant match	< 0.700
CRC21.h	EU564004	374	98	EF406903	Uncultured bacterium clone DSS_7DAYS_ C05_T7P	Mouse colon	No significant match	< 0.700
CRG4.C	BJ563981	337	98	EF401722	Uncultured bacterial clone	Human fecal sample	No significant match	< 0.700
BL55.b	BJ563972	358	98	AB200871.1	Streptococcus thermophilus strain:DL1	A Georgia yogurt	Streptococcus thermophilus	0.916	AY687383
BL59.a	BJ563974	358	98	AY762259.1	Streptococcus iniae strain SCCF5L	strain=”SQCF5L	Streptococcus uberis (T); ATCC 19436	0.936	AB002526
C13.b	BJ563993	376	99	AM 157442	Str. pneumonieae 4V4	Human breast milk	Streptococcus pneumoniae (T); ATCC 33400	0.948	AF003930
CRC16.d	BJ563988	374	99	AY138233	Uncultured Streptococcus	Human estomach biopsy	Streptococcus uberis (T); ATCC 19436	0.917	AB002526
CRC16.f	BJ563990	376	99	AY959090	Uncultured bacterium clone rRNA317	Human vaginal epithelium	Streptococcus intermedius (T); ATCC27335	0.952	AF104671
C19.c	BJ563996	375	99	EF405528	Uncultured bacterium clone SJTU_G_01_33	Human fecal sample	Clostridium orbiscindens (T) ; DSM 6740	0.910	Y18187
CRC21.b	BJ563998	378	99	EF404797	Uncultured bacterium clone SJTU_C_10_72	Human fecal sample	No significant match	< 0.700
BL59.C	BJ563976	351	99	AM157442.1	Streptococcus pneumoniae clone 4V4
BL59.d	BJ563977	357	99	AM 157442.1	Streptococcus pneumoniae clone 4V4	Breast milk	Streptococcus pneumoniae R 6	0.944	AED08537
BL60.a	BJ563973	355	99	AM 157442.1	Streptococcus pneumoniae clone 4V4	Breast milk	Streptococcus pneumoniae 4V4
BL55.C	BJ563971	356	100	AM157442.1	Streptococcus pneumoniae clone 4V4	Breast milk	Streptococcus pneumoniae 4V4	0.989	AM 157442

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