Small plasmids in Streptococcus dysgalactiae subsp. equisimilis isolated from human infections in southern India and sequence analysis of two novel plasmids

International Journal of Medical Microbiology 305 (2015) 365–369

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Small plasmids in Streptococcus dysgalactiae subsp. equisimilis isolated from human infections in southern India and sequence analysis of two novel plasmids René Bergmann ∗ , D. Patric Nitsche-Schmitz Department of Medical Microbiology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany

a r t i c l e

i n f o

Article history: Received 3 September 2014 Received in revised form 6 January 2015 Accepted 9 February 2015 Keywords: Streptococcus Plasmid Bacteriocin emm Gene

a b s t r a c t Small plasmids are frequently found in S. pyogenes isolates from human infections in India. Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a streptococcal subspecies that is genetically similar to S. pyogenes and has a similar ecology. Therefore, we determined the distribution of small plasmids in a collection of 254 SDSE isolates, comprising 44 different emm-types and emm non-typable strains, from southern India, utilizing an established PCR based method. Briefly, 1.2% (n = 3) of the isolates were positive for repA (encoding the replication initiation protein A) and 1.6% (n = 4) were repB positive (encoding the replication initiation protein B). One isolate (G315) showed a co-detection of repB and dysA (encoding the bacteriocin dysgalacticin) which is characteristic for previously described pDN281/pW2580-like plasmids, observed in SDSE and S. pyogenes. The remaining plasmid bearing isolates showed no characteristic co-detection of known plasmid-associated genes. Thus, plasmids pG271 and pG279, representatives for repB and repA harboring plasmids, respectively, were analyzed. The plasmids pG271 and pG279 could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. Like the characterized small native plasmids of S. pyogenes from India, the SDSE plasmids discovered and described in this study did not carry any of the known antibiotic resistance genes. SDSE bore less of the investigated small native plasmids that were distinct from the small native plasmids of S. pyogenes of the same geographic region. This indicates a low rate of lateral transfer of these genetic elements between these two related streptococcal species. © 2015 Published by Elsevier GmbH.

Introduction Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a ␤hemolytic Gram positive bacterium of the pyogenic group of streptococci. The vast majority of SDSE carry the Lancefield group G carbohydrate on their surface, while another considerable portion belongs to Lancefield group C (Reißmann et al., 2010; McDonald et al., 2007; Loubinoux et al., 2013). Rarely, SDSE possess group A or L antigens (Tanaka et al., 2008). As the related Streptococcus pyogenes of Lancefield group A (Jensen and Kilian, 2012), SDSE carry an emm gene that codes for the anti-phagocytic M protein (Reißmann et al., 2010; Simpson et al., 1987). Variability in the 5 end of the emm gene is exploited in genotyping of S. pyogenes and SDSE isolates (Reißmann et al., 2010; Facklam et al., 1999). These two species share several virulence factors in addition to the M

∗ Corresponding author. Tel.: +49 531 6181 4504; fax: +49 531 6181 4499. E-mail address: [email protected] (R. Bergmann). http://dx.doi.org/10.1016/j.ijmm.2015.02.004 1438-4221/© 2015 Published by Elsevier GmbH.

protein (Cleary et al., 1991; Davies et al., 2007a) and cause a similar spectrum of diseases that comprises pharyngitis, pyoderma, urinary tract infections, necrotizing fasciitis, septicemia and streptococcal toxic shock syndrome (Reißmann et al., 2010; Nohlgard et al., 1992; Brandt and Spellerberg, 2009; Baracco and Bisno, 2006). In contrast to Europe and North America, India and other geographic regions reported high colonization rates for SDSE and high incidence rates for SDSE infections (McDonald et al., 2007; Ogunbi et al., 1978; Brahmadathan and Koshi, 1989; Bramhachari et al., 2010; Broyles et al., 2009). Information about the factors that lead to these differences in the epidemiology of SDSE is scant. Interspecies horizontal gene transfer between SDSE and other species of the pyogenic group, S. agalactiae and S. pyogenes has been reported (Davies et al., 2005, 2007a,b, 2009). Exogenous genetic elements involved in this transfer comprise prophages, integrated conjugative elements and plasmids that transmit antibiotic resistance genes and bacteriocins, thereby contributing to fitness of the recipient strain (Beres and Musser, 2007; Banks et al., 2002). To date, only two small SDSE plasmids (<7 kb) are

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known and sequenced. One plasmid is pW2580, which encodes the bacteriocin dysgalacticin (Heng et al., 2006). An almost identical plasmid (99% identity), designated pDN281, was also found in S. pyogenes (GenBank: AY995189). The second SDSE plasmid p5580 is almost identical to the non-self-transmissible erm(T)-carrying plasmids of S. pyogenes (pRW35: 99% identity) and S. agalactiae (pGB2001: 99% identity) and confers resistance to erythromycin to all the three species (Palmieri et al., 2013; Woodbury et al., 2008; DiPersio et al., 2011). For S. pyogenes three additional plasmids were described: pDN571, which encodes the bacteriocin streptococcin A-M57 (Heng et al., 2006); pA852 and pA996, both encoding hypothetical bacteriocins (Bergmann et al., 2013). Recently, we analyzed 279 S. pyogenes isolates from India for the presence of the above mentioned plasmids and observed that 21% of the isolates harbored a small natural plasmid (Bergmann et al., 2013) that could contribute to the prevalence of certain emm-types. We have now examined a collection of SDSE isolated from human infections in Vellore, southern India. We investigated these isolates for the presence and distribution of small natural plasmids that may contribute to the prevalence of certain genotypes, and high incidence of SDSE infections in this particular region.

Biosystems) and an ABI 3730XL capillary sequencer. Sequences were edited and assembled using the GAP4 program (Staden, 1996). Nucleotide sequence accession numbers The nucleotide sequence data reported here have been deposited in GenBank nucleotide sequence database under the accession numbers KJ951052 (plasmid pG271) and KJ951051 (plasmid pG279). Computational analysis Open reading frames (ORFs) of the plasmid sequences were predicted using GLIMMER (Delcher et al., 1999). Amino acid sequences of the predicted ORFs were analyzed by blastp and signal peptide predictions were performed with SignalP 4.1 (Petersen et al., 2011). Results Detection of plasmid specific sequences

Plasmids were amplified in a reaction volume of 50 ␮l containing 0.2 ␮M of suitable primers (supplemental Table S3), 0.2 mM of each dNTP, 1× Phusion HF buffer, 1 unit of Phusion high-fidelity DNA polymerase (New England Biolabs) and 2 ␮l of genomic DNA (100–400 ng). The amplification conditions were: denaturation at 98 ◦ C for 30 s; 27 cycles of denaturation at 98 ◦ C for 15 s, annealing at gene-specific temperatures (supplemental Table S3) for 30 s, and elongation at 72 ◦ C for 30 s per 1 kb; final elongation at 72 ◦ C for 10 min. The PCR products were separated by agarose gel electrophoresis.

The distribution of small native plasmids in a previously characterized collection of 254 SDSE isolates from southern India, comprising 44 different emm-types and emm non-typable strains (Reißmann et al., 2010) was examined by an established PCR based method (Bergmann et al., 2013). The PCRs detect plasmid replication initiation protein genes repA, repB, rep2 and a repB homologue that was found in S. pyogenes (Bergmann et al., 2013) and is herein referred to as repB . Furthermore, all isolates were analyzed for the presence of the plasmid associated genes ermT (macrolide resistance gene), scnM57 (encoding the bacteriocin streptococcin M57) and dysA (encoding the bacteriocin dysgalacticin). In detail, 1.2% (n = 3) of the 254 SDSE isolates from southern India were positive for repA (isolates G190, G193 and G279) and 1.6% (n = 4) were repB positive (isolates C118, G271, G315 and G332) (Table 1). Genes rep2 and repB , which are indicative for pRW35- and pA852-like plasmids, respectively, were not detected. Moreover, the genes ermT and scnM57, indicative for pRW35- and pDN571-like plasmids, respectively, were not detected. The repBpositive isolate G315 was the only one that bore dysA, thus showing the combination that is characteristic for pDN281/pW2580-like plasmids (Beres and Musser, 2007; Woodbury et al., 2008) (Table 2). The remaining isolates that were positive in the PCR for small plasmids, were negative for pA996 ORF5 and pA852 ORF3. Both pA996 ORF5 and pA852 ORF3 encode putative bacteriocins in S. pyogenes (Bergmann et al., 2013) (Table 1). For further examination repB-positive plasmids were amplified by inverted PCR using primers rep02B fwd and rep02 rev (supplemental Table S3). Isolate G315 produced an amplicon of 3 kbp (Table 1), which is similar to the size of known pDN281/pW2580-like plasmids (Bergmann et al., 2013). For the remaining repB-positive isolates C118, G271 and G332, the inverted PCR amplified plasmid sequences with a size of 6.5 kbp (Table 1). Inverted PCR with primers pG279 f1 and pG279 r1 amplified 2.0 kbp plasmids from the three repA-positive isolates G190, G193 and G279 (Table 1). Taken together, a pDN281/pW2580-like plasmid and hitherto uncharacterized repA- and repB-positive plasmids were detected in our collection of SDSE from southern India. All uncharacterized repA- and repB-positive plasmids were sequenced and analyzed. Representative plasmids of isolates G271 and G279 are described below in detail.

Sequencing of plasmids

Sequence analysis of plasmid pG271

Cycle sequencing was performed using the ABI PRISM BigDye Terminator v 3.1. Ready Reaction Cycle Sequencing Kit (Applied

Plasmid pG271, which derived from an SDSE isolate of emm type stG652.0, had a total size of 6436 bp with a GC content of 33.8%.

Material and methods Bacterial strains S. dysgalactiae subsp. equisimilis isolates from human infections were collected at the Christian Medical College, Vellore. The collection comprised 254 S. dysgalactiae subsp. equisimilis isolates from different suppurative foci and invasive infections (supplemental Tables S1 and S2). Genomic DNA was isolated by using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer’s protocol with a minor variation: the incubation with proteinase K was carried out at 70 ◦ C for 30 min. Emm-type analysis of the collection was described previously (Reissmann et al., 2010). PCR screening for plasmids PCR screening was performed in a reaction volume of 25 ␮l containing 0.2 ␮M of suitable primers (supplemental Table S3), 0.2 mM of each dNTP, 1× CoralLoad PCR Buffer, 1.5 mM MgCl2 , 0.04 unit of Taq DNA polymerase (QIAGEN, Hilden, Germany) and 1 ␮l of genomic DNA (50–200 ng). After initial denaturation at 94 ◦ C for 4 min, 25 cycles of denaturation at 94 ◦ C for 1 min, annealing at gene-specific temperatures (supplemental Table S3) for 30 s and an extension phase at 72 ◦ C for 1 min per kb of gene length, the cycle reaction was terminated by elongation at 72 ◦ C for 5 min. PCR products were analyzed by agarose gel electrophoresis. Amplification of plasmids

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Table 1 Streptococcus dysgalactiae subsp. equisimilis isolates harboring a plasmid A collection of 254 SDSE isolates was analyzed for the presence of plasmid specific genes. Plasmids were detected in five different emm-types. Two isolates of emm-type stG652 were tested positive one harboring a repA, the other one a repB carrying plasmid. Plasmid harboring isolate

emm Type

G190 G193 G279 C118 G271 G332 G315

stG652.1 stC1400.2 stG6.0 stG485.0 stG652.0 stG485.0 stG6792.3

Approx. plasmid size (bp)

Plasmid family

2000 2000 2000 6500 6500 6500 3000

pC194/pUB110

pMV158

pMV158

Plasmid specific gene repA

repB

rep2

repB

dysA

scnM57

ermT

pA852 ORF3

pA996 ORF5

+ + + − − − −

− − − + + + +

− − − − − − −

− − − − − − −

− − − − − − +

− − − − − − −

− − − − − − −

− − − − − − −

− − − − − − −

Table 2 Distribution of plasmids in S. pyogenes and SDSE from India. Species

Region

*1

S. pyogenes

SDSE Plasmid family *1

Northern India Southern India Total Southern India

No. isolates

96 183 279 254

Total plasmid positive

21.9% (n = 21) 20.2% (n = 37) 20.8% (n = 58) 2.8 (n = 7)

Prototype plasmids with characteristic gene(s) pDN571

pA996

pG279

pW2580/pDN281

pG271

pA852

repA scnM57

repA pA996 ORF5

repA

repB dysA

repB

repB pA852 ORF3

0 0 0 1.2% (n = 3)

3.1% (n = 3) 3.8% (n = 7) 3.6% (n = 10) 0.4% (n = 1) pMV158

0 0 0 1.2% (n = 3)

9.4% (n = 9) 9.3% (n = 17) 9.3% (n = 26) 0

1.0% (n = 1) 8.3% (n = 8) 3.8% (n = 7) 3.3% (n = 6) 2.9% (n = 8) 5.0% (n = 14) 0 0 pC194/pUB110

Woodbury et al. (2008).

Ten ORFs encoding putative proteins were predicted (supplemental Table S4, Fig. 1). The amino acid sequence of the predicted RepB protein (pG271 ORF1), was 99% similar to RepB proteins of the three pMV158 family of rolling-circle plasmids (RCP) pW2580 (Heng et al., 2006) and pSdyT132 (GenBank: DQ173493.1) of SDSE and pDN281 (GenBank: AY995189.1) of S. pyogenes (supplemental Table S4). The predicted single-strand origin of replication of pG271 contained the sequence 5 -TTTATGCCGAGAAA-3 that was almost identical to the 14 bp recombination site B (RSB ) 5 TTTATGCCGTGAAA-3 (del Solar et al., 1998). The differing single nucleotide is underlined. Based on this homology the single-strand origin of replication (sso) of pG271 was assigned to type ssoA. The predicted double-strand origin of replication bore the nick sequence (nic) 5 -TACTACG/A-3 , which is conserved in all plasmids

Fig. 1. Circular map of plasmid pG271. The 5 -end of the replication initiation protein gene repB was defined as position 1 of the plasmid sequence. Ten predicted ORFs and their transcriptional orientations are shown as black arrows. The doublestrand origin of replication (dso) and the single-strand origin of replication (sso) are depicted as gray boxes.

of the pMV158 family except of pKMK1(del Solar et al., 1993). The binding region bind is located 69 nucleotides downstream from the nic region. It contains three directly repeated iterons, two identical copies of the sequence 5 -TCGGCGAGTTT-3 and one similar sequence 5 -TCGGTTGCGAGTTT-3 . The two identical copies of the pG271 iterons showed a high degree of nucleotide sequence identity to the three directly repeated iterons in plasmid pLS1, in which they serve as the binding site of the Rep protein (de la Campa et al., 1990). pG271 ORF10 coded for the transcriptional repressor protein CopG that was identical to CopG of the SDSE plasmids pW2580 and pSdyT132 and the S. pyogenes plasmid pDN281. This element is specific for the streptococcal pMV158 plasmid family and controls the plasmid copy number during bacterial proliferation (del Solar et al., 1990). Proteins encoded by pG271 ORF4 and pG271 ORF5 were 100% and 98% similar (100% and 96% identical) to hypothetical proteins of plasmid pSdyT132 (GenBank: DQ173493.1). Homology search suggested that pG271 ORF4 encodes an antitoxin component of the prevent-host-death YefM superfamily proteins (Anantharaman and Aravind, 2003), while pG271 ORF5 is predicted to encode a toxin that is essential for plasmid maintenance (Roberts and Helinski, 1992). pG271 ORF2 encoded a hypothetical protein of 57 aa with 95% similarity (93% identity) to the dysgalacticin immunity factor of plasmid pW2580 (Swe et al., 2010). However, in contrast to pW2580, pG271 lacked a dysgalacticin gene. The remaining hypothetical proteins, encoded by pG271 ORF6, pG271 ORF7, pG271 ORF8 and pG271 ORF9 were 60–84% similar to hypothetical proteins of unknown functions in the GenBank database. Only pG271 ORF3 was predicted to encode a protein with signal peptide sequence for export by a Sec-dependent transport system and would yield a 21 kDa extracellular protein after processing by signal peptidase. The sequence of this hypothetical protein did not show sufficient homology to other proteins that would allow functional predictions. Like SDSE isolate G271, isolates C118 and G332 harbored a 6.5 kbp plasmid. Sequence comparison of these repB-positive (dysA-negative) SDSE plasmids revealed nucleotide identity of 98.8% between pG271 and pC118, 99.3% between pG271 and pG332 and 99.5% between pG332 and pC118.

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Fig. 2. Circular map of plasmid pG279. The 5 -end of the replication initiation protein A gene repA was chosen as position 1 of the plasmid sequence. Three predicted ORFs and their transcriptional orientations are shown as black arrows. The doublestrand origin of replication (dso) and the single-strand origin of replication (sso) are depicted as gray boxes.

Sequence analysis of plasmid pG279 Sequencing of plasmid pG279 from SDSE isolate G279 (emm type stG6.0) revealed its length of 1940 bp and GC content of 35.1%. Three ORFs encoding putative proteins were predicted (supplemental Table S5, Fig. 2). pG279 ORF1 encoded a hypothetical protein of 314 amino acids that showed 95% similarity (91% identity) with the RepA protein of plasmid pDN571 of S. pyogenes (Heng et al., 2004), a plasmid of the pC194/pUB110 RCP family (supplemental Table S5). pG279 ORF2 and pG279 ORF3 coded for hypothetical proteins of 48 and 52 amino acids, respectively. BlastP analysis revealed no significant similarity of these two proteins to other proteins of the GenBank database (supplemental Table S5). The sso of pG279 was 100% identical to the sso of pW2580 (Heng et al., 2006) and 99% identical to the sso of pSdyT132 (GenBank: DQ173493.1). Both pW2580 and pSdyT132 were found in SDSE. Moreover, the sso of pG279 showed 99% and 97% identity to the sso of the S. pyogenes plasmids pDN281 (GenBank: AY995189.1) and pDN571 (Heng et al., 2004), respectively (supplemental Table S5). The putative dso is identical to the dso of plasmid pDN571 and bore the heptameric potential nick site 5 -CTTGATA-3 . This sequence is conserved in most of the plasmids of the pC194/pUB110 RCP family (Seery et al., 1993). Like SDSE isolate G279, isolates G190 and G193 harbored a 2 kbp repA-positive plasmid. Sequence comparison of these SDSE plasmids revealed nucleotide identity of 99.8% between pG279 and pG190 or pG193, and 99.9% between pG190 and pG193. Discussion A PCR based method was utilized in this study to detect small plasmids that bear the plasmid specific sequences ermT (present in S. pyogenes (Woodbury et al., 2008), SDSE (Palmieri et al., 2013) and S. agalactiae (DiPersio et al., 2011)), dysA (present in S. pyogenes (GenBank: AY995189) and SDSE (Heng et al., 2006)), scnM57 (present in S. pyogenes (Heng et al., 2004)), pA996 ORF5, pA852 ORF3 (both present in S. pyogenes (Bergmann et al., 2013)) or one of the replication initiation protein genes rep2 (present in S. pyogenes (Woodbury et al., 2008), SDSE (Palmieri et al., 2013) and S. agalactiae (DiPersio et al., 2011)), repA (present in S. pyogenes (Bergmann et al., 2013; Heng et al., 2004)), repB (present in S.

pyogenes (Bergmann et al., 2013) and SDSE (Heng et al., 2006)) or repB (present in S. pyogenes (Bergmann et al., 2013)). Thus, our study is limited to plasmid associated factors that are known to occur in the species S. pyogenes, SDSE and S. agalactiae, which all belong to the pyogenic group of streptococci. Presence of additional small plasmids in SDSE, which did not bear homologous rep genes, cannot be excluded. Like the known small native plasmids of S. pyogenes from India, the plasmids characterized in this study did not carry any of the known antibiotic resistance genes. In S. pyogenes macrolide resistance genes were found on larger plasmids (>7 kbp) that bear repS (Liu et al., 2007; Ceglowski and Alonso, 1994), which were not investigated in this study but may occur in the SDSE isolates from southern India. Typically located on plasmids or transposons (Varaldo et al., 2009) ermA or ermB were detected in 2.4% of these isolates (data not shown). With a length of 6436 bp, the newly characterized pMV158 family of rolling circle plasmid pG271 is larger than all previously described S. pyogenes or SDSE plasmids that bore one of the replication initiation protein genes listed above (Bergmann et al., 2013). Bearing dysI but lacking dysA, pG271 codes only for the immunity factor of a toxin/anti-toxin system that is based on the extracellular bacteriocin dysgalacticin and may be involved in plasmid stability and/or bacterial competition (Heng et al., 2006; Swe et al., 2009, 2010). Lack of dysA may have resulted in a lack of function in plasmid stabilization that may be compensated by pG271 ORF4 and pG271 ORF5, which code for a potential plasmid stabilization system. Moreover, plasmid pG271 contains 5 ORFs that encode hypothetical proteins with lengths between 80 and 264 amino acids and unknown function (supplemental Table S4). pG271 ORF3 was predicted to encode a signal peptide sequence for extracellular secretion of a mature 21 kDa protein. The hypothetical protein is not homologous to other proteins, which impedes functional predictions. Functional investigations could clarify if this protein compensates for a lack of the extracellular function of dysgalacticin in bacterial competition. Plasmids from isolates C118 and G332 were nearly identical to pG271. Three plasmids with an approximate size of 2 kbp carried repA. Sequence analysis revealed high nucleotide identity between the plasmids and identified them as plasmids of the pC194/pUB110 family (Table 1). To our knowledge these are the first plasmids of this family that were found in SDSE. Their smaller size (<3 kpb) and two unique ORFs distinguish the SDSE plasmids from previously described S. pyogenes plasmids that were abundant in India (Fig. 2; Supplemental Table S5). Frequently occurring in S. pyogenes isolates from southern India (n/N = 7/183; 3.6%) (Bergmann et al., 2013), where it was present in 19% (n/N = 7/37) of all detected plasmids, dysA was found in only one of the SDSE isolates (G315) from this geographic region (n/N = 1/254; 0.4%) (Table 2). The co-detection of repB and dysA, originally described for plasmid pW2580 from SDSE (Heng et al., 2006), identified the plasmid in G315 as a pDN281/pW2580-like plasmid, which are all members of the pMV158 plasmid family (Tables 1 and 2). In a previous study, small native plasmids were detected in about 21% (n = 58) of S. pyogenes isolates from India (N = 279). Of these plasmids, 45% (n = 26) were positive for repB , 38% (n = 22) positive for repA and 17% (n = 10) positive for repB. A similar rate of S. pyogenes isolates with small native plasmids was determined for southern India alone (20%; n/N = 37/183) (Table 2). A comparatively low rate of small native plasmids was detected in SDSE isolates from southern India, where 3% (n = 7) of 254 isolates were tested positive for repA (n = 3) or repB (n = 4). Co-detection of the two genes did not occur (Table 1). Despite considerable genetic similarity to S. pyogenes and a similar ecology, we detected a lower rate of the investigated small native plasmids in SDSE from southern India as compared to S. pyogenes. Small plasmids that were

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