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Skin and osteoarticular infections (SKI and OAI, respectively) account for almost one-third of Streptococcus agalactiae infections in nonpregnant adults. We evaluated the genetic diversity and phylogeny of 58 S. agalactiae strains responsible for adult SKI or OAI and of 61 S. agalactiae strains from cases of adult human colonization (HCol) by serotyping and multilocus sequence typing (MLST). We also assessed the prophage DNA content of the genomes of these strains by a PCR-based method. We found that 63% of SKI and 56% of OAI occurred in people aged 55 years and over. Overall, 71% of SKI strains were of serotype Ia or V, and 91% of OAI strains were of serotype Ia, III, or V. Strains of clonal complexes 1 and 23 (CC1 and CC23) were associated with 79% of SKI cases and 62% of OAI cases. Seven groups of strains, groups A, B, C, D, E, F, and G, were obtained by performing a hierarchical analysis on the basis of prophage DNA-PCR data. We found that 85% of CC1 strains clustered in DNA prophage group D, the group with the highest prophage DNA content (average, 4.4; average of absolute deviations [AVEDEV], 0.9). The CC23 strains displayed the greatest diversity in prophage DNA fragment content, but 47% of CC23 strains clustered in group B, which also had a high average prophage DNA content per strain (average, 2.3; AVEDEV, 0.6). Many (65%) of the OAI strains were in prophage DNA group D, whereas 83% of the SKI strains were in prophage DNA groups B and D. These data suggest that S. agalactiae strains from CC1 and CC23 may be subject to particular transduction mechanisms in gene recombination, rendering them particularly capable of invading the skin, bone, or joints in adults.
Streptococcus agalactiae was initially described in 1887 as an animal pathogen causing bovine mastitis (36). Since the 1960s, when human vaginal carriage of S. agalactiae was first documented, S. agalactiae has frequently been linked to neonatal infections and this bacterium has become the leading neonatal pathogen in developed countries (10, 13, 22, 23, 27, 34). S. agalactiae was rarely isolated from nonpregnant adults until 2 decades ago, when such infections began to be reported, particularly for the elderly and for individuals with underlying conditions such as diabetes mellitus, cancer, and a compromised immune system (4, 15, 37, 40-42). However, such infections have been reported even for adults without a known susceptibility factor (29, 33). Many case reports of clinical skin and osteoarticular infections (SKI and OAI, respectively) due to S. agalactiae in adults have been published in recent years, with these infections accounting for at least one-third of the reported cases of S. agalactiae infection in adults (41, 42).
Many genetic markers have been identified as associated with S. agalactiae clones specifically responsible for meningitis in neonates. Indeed, most of the S. agalactiae strains isolated from the cerebrospinal fluid of neonates belong to clonal complex 17 (CC17) and have particular mobile genetic elements, such as the group II intron GBSi1 (2) and particular prophage DNA fragments (47). No particular markers or virulence factors of S. agalactiae strains have been associated with any other disease.
Phages are important vehicles for horizontal gene exchange within bacterial populations and account for much of the genomic variation observed within bacterial species (8, 11, 28). Temperate phages affect bacterial fitness by modifying anchor points for genomic rearrangements, by disrupting genes, by protecting against lytic infection, by lysing competing strains through prophage induction, and by introducing new fitness factors (8, 19). Prophage acquisition, accounting for much of the molecular diversity of the Streptococcus pyogenes genome, rendered some of the strains of this species virulent through the acquisition of phage-encoded virulence factors and enhanced pathogen survival by improving resistance to host defenses under certain circumstances (1, 9). Little is currently known about S. agalactiae phages. They were first isolated in 1969 (39), and more-recent analyses of sequenced S. agalactiae strains have revealed the presence of abundant regions resembling prophages (16, 44, 45). We recently induced phages from S. agalactiae strains of various phylogenetic lineages, characterized them molecularly, and determined their lytic activities (12). The various molecular phage groups were found to correspond to particular strain lineages, with specific morphological features and lytic activities, suggesting a role for phage-mediated horizontal gene transfer in the evolution of the species and the emergence of lineages with a more specific role in particular diseases.
In this study, we characterized S. agalactiae strains isolated from skin and osteoarticular infections in adults, using serotyping and multilocus sequence typing (MLST) to determine the phylogenetic relationships and molecular features of the strains involved through comparison with the characteristics of strains involved in human colonization (HCol). We used a PCR-based method recognizing S. agalactiae prophages to determine the prophage content of strain genomes (12). The genetic relationships between prophage DNA regions of strains were determined by hierarchical analysis. Correlations between the prophage DNA content, the clinical circumstances of isolation, and the phylogenetic position of S. agalactiae strains were investigated.
We studied 119 strains of S. agalactiae. We isolated 24 strains from samples taken from patients with the following skin infections (SKI): whitlows (seven cases), perforating ulcers of the foot (seven cases), cellulitis (two cases), erysipelas (three cases), and cutaneous abscesses (five cases). We studied 34 strains responsible for osteoarticular infections (OAI), isolated from joint fluids in 26 cases and from bone biopsy specimens in 8 cases. All strains were isolated from nonpregnant adult patients (≥18 years of age) admitted to hospitals in various regions of France from 2002 to 2007. We characterized 61 human colonization (HCol) strains previously isolated from nonpregnant adults (48) by the same methods to allow a comparison of the molecular characteristics between these strains and those responsible for SKI and OAI.
Strains were serotyped by PCR, as previously described (26).
The strains were analyzed by MLST, as described by Jones et al. (24). Strains were grouped into clonal complexes (CCs) with the eBURST software program (http://eburst.mlst.net/). A neighbor-joining tree was generated from allelic profile data by Phylodendron (http://pubmlst.org/).
We previously identified and characterized bacteriophages and prophage remnants from S. agalactiae strain genomes and designed primer pairs recognizing the prophage sequences of S. agalactiae bacteriophages for use in PCR (12, 47). Ten of these primer pairs were used here for the evaluation, by PCR, of the prophage content of S. agalactiae strains (Table (Table1).1). PCR was carried out with a Chromo 4 system instrument (Bio-Rad, Hercules, CA) on DNA isolated from the strains. PCR was carried out in a final volume of 25 μl, containing 5 μl of extracted DNA, a 0.5 μM concentration of each primer, and 1× iQ SYBR green Supermix (Qiagen SA, Courtaboeuf, France) including 3 mM MgCl2. Amplification was performed over 40 cycles of 10 s at 94°C, 10 s at the annealing temperature (45°C for F5, 48°C for F7, 49°C for F10 and SAK_2094, 50.5°C for SAJ_2395 and SAK_1326, 52°C for SAK_0748, or 54°C for SAG0566, SAK_2090, and SAK_0738), and 30 s at 72°C. The reaction products were then cooled to 35°C and subjected to a post-PCR melting cycle by increasing the temperature by 0.2°C for each 10-s cycle, up to 95°C.
The genetic relationships between the prophage DNA regions of strain genomes were investigated by a hierarchical analysis based on the Jaccard dichotomy coefficient method, as implemented in the SYSTAT 12 software program. The genetic feature analyzed was the presence of a PCR amplicon corresponding to the prophage sequences studied. An absence of gene amplification was not considered to indicate similarity between the studied strains.
Data were analyzed by chi-square tests and Fisher's exact tests to evaluate associations, with a P value of ≤0.05 considered significant.
S. agalactiae skin infections (SKI) were equally distributed between the sexes, like S. agalactiae human colonization (HCol) (Table (Table2).2). In contrast, S. agalactiae osteoarticular infections (OAI) were significantly more frequent (P = 0.02) (Table (Table2)2) in men (25/34; 74%) than in women (9/34; 26%), whereas no such difference between the sexes was observed for colonization in men (29/61; 48%) and in women (32/61; 52%).
S. agalactiae HCol strains were most frequently isolated from individuals under the age of 55 years (44/61; 72%), whereas S. agalactiae SKI and OAI strains were most frequently isolated from individuals aged 55 years and over (15/24 [63%] and 19/34 [56%], respectively; P = 0.003) (Table (Table22).
All S. agalactiae strains responsible for SKI or OAI could be serotyped, whereas 7 of the 61 HCol strains (11%) could not be typed (Table (Table2).2). More than two-thirds of the strains responsible for SKI (71%) belonged to serotypes Ia (38%) and V (33%), and most of the strains isolated from OAI (91%) were of serotype V (38%), serotype III (29%), or serotype Ia (24%). In contrast, HCol strains were more equally distributed between the various serotypes (5 to 21%) (Table (Table22).
We identified 31 different sequence types (STs) among the 119 strains tested. The eBURST software program assigned 114 strains from 28 of the 31 STs to seven clonal complexes (CCs), CC1, CC7, CC8, CC17, CC19, CC23, and CC388 (Table (Table3).3). The genetic relationship between the STs and CC is presented as a dendrogram (Fig. (Fig.11).
HCol strains were more genetically diverse than the strains responsible for infections; the 61 HCol strains belonged to 23 different STs, with a maximum of 9 strains for a single ST (15%, for ST-1) (Table (Table33).
The distributions of SKI and HCol strains between the various STs (Table (Table3)3) differed significantly (P < 0.00001). SKI strains were less diverse, with the 24 strains belonging to only 10 STs. SKI strains were more frequently classified as ST-1 (7/24; 29%) or ST-23 (7/24; 29%) than were HCol strains (9/61 [15%] ST-1 and 5/61 [8%] ST-23). Similarly, the distributions of SKI and HCol strains between the various CCs differed significantly (P < 0.00001) (Table (Table3).3). SKI strains were more likely to belong to CC1 or CC23 (19/24; 79%) than HCol strains (30/61; 49%).
The distributions of OAI and HCol strains between the various STs (Table (Table3)3) differed significantly (P < 0.00001). The 34 OAI isolates belonged to 14 STs. OAI strains were more frequently of ST-1 (11/34; 32%) and ST-23 (6/34; 18%) than were HCol strains (9/61 [15%] ST-1 and 5/61 [8%] ST-23). Nevertheless, the distributions of OAI strains and of HCol strains between CCs did not differ significantly (P = 0.2), although OAI strains were more likely to belong to CC1 or CC23 (21/34; 62%) than HCol strains (30/61; 49%).
S. agalactiae strains of each serotype were distributed between several STs (Table (Table3),3), but the strains of serotypes Ia, Ib, IV, and V were mostly of ST-23 (50%), ST-8 (53%), ST-196 (67%), and ST-1 (70%), respectively. Serotype Ia, Ib, and V isolates belonged principally to CC23 (86%), CC8 (73%), and CC1 (73%), respectively.
The prophage DNA fragments studied here were not detected by PCR in four S. agalactiae strains, all isolated from HCol cases. For each of the remaining 115 strains, PCR amplified 1 to 7 of the 10 prophage DNA fragments studied. The genetic relationships between the prophage DNA regions of strain genomes were represented as a dendrogram (Fig. (Fig.2).2). This analysis assigned the strains to seven major prophage DNA groups, A to G. The natures and frequencies of the prophage DNA fragments amplified from the strains differed significantly between prophage DNA groups (Table (Table4)4) (P < 0.00001). Three different patterns of prophage DNA fragment amplification were observed in these groups (Table (Table4).4). For the 49 strains of prophage group D, all the prophage targets studied were found in at least one strain and PCR amplified a large number of prophage DNA fragments in each strain (average, 4.4) (Fig. (Fig.2).2). For the 24 strains of prophage group B, a large number of prophage DNA fragments were amplified (only one prophage sequence was never amplified [SAK_0748]) and the mean number of prophage DNA fragments amplified per strain was 2.3 (Fig. (Fig.2).2). Only one or two prophage DNA fragments were amplified for 35 strains from the 42 strains of the other five prophage DNA groups (A, C, E, F, and G). In each of these groups, the mean number of prophage DNA fragments amplified per strain was ≤2 (Fig. (Fig.22).
Strains of the three different clinical origins (SKI, OAI, and HCol) were differentially distributed between prophage DNA groups A to G (Table (Table4)4) (P = 0.003). HCol strains were evenly distributed between prophage DNA groups A to F, whereas 65% of OAI strains (22/34 strains) (Table (Table4)4) belonged to prophage DNA group D, the members of which had the largest numbers of amplified prophage DNA fragments. Similarly, 83% of SKI strains (20/24 strains) (Table (Table4)4) belonged to prophage DNA groups B and D, which displayed the highest numbers of amplified prophage DNA fragments. In contrast, 17 of the 18 strains of prophage groups A, E, and F, in which the number of amplified prophage DNA fragments was low, were HCol strains.
The distribution of strains from the various serotypes, STs, and CCs between prophage DNA groups was not random (Table (Table4)4) (P < 0.00001). Strains of the two major lineages, ST-1 and ST-23, and of the corresponding clonal complexes, CC1 and CC23, which were frequently implicated in SKI and OAI, had particular characteristics in terms of their prophage DNA content. All 27 ST-1 strains (100%) and 34 of the 40 CC1 strains (85%) (Table (Table4),4), most of which were of serotype V (Fig. (Fig.2),2), belonged to prophage DNA group D. And the six CC1 strains that did not belong to prophage DNA group D were rarely isolated from patients with infectious disease (only one strain, from a case of OAI) (Fig. (Fig.2).2). The CC8 and CC19 strains mostly belonged to prophage groups C (9/18; 50%) and B (7/11; 64%), respectively (Table (Table44 and Fig. Fig.2).2). The strains of phylogenetic lineage ST-23 and its corresponding clonal complex, CC23, were mostly of serotype Ia and were distributed between five of the seven prophage DNA groups (groups B, C, D, E, and G) (Fig. (Fig.22 and Table Table4).4). Therefore, they displayed considerable diversity in terms of the prophage DNA fragments amplified. Nevertheless, 14 of the 30 strains (47%) of CC23 belonged to prophage DNA group B, which included 64% of the CC19 strains.
S. agalactiae infections in nonpregnant adults have greatly increased in frequency over the last 2 decades in the United States and Europe (4, 15, 37, 40). Many clinical cases of S. agalactiae SKI and OAI have been reported. Our data confirm that S. agalactiae SKI and OAI are significantly more frequent in older people (≥55 years of age) (14, 15, 40), that most of the strains responsible for SKI (71%) belong to serotypes Ia and V, and that serotypes V, III, and Ia predominate among OAI strains (91%) (5, 14, 18, 46). Nevertheless, no other study to date has specifically focused on molecular characterization, including evaluation of the prophage content of S. agalactiae strains responsible for SKI and OAI.
Only strains from a particular phylogenetic lineage, initially recognized by multilocus enzyme electrophoresis (MLEE) (35, 38) and more recently defined as ST-17 by MLST, have been shown to be associated with a particular disease, due to a higher likelihood of their invading the central nervous system (CNS) of neonates (3, 24, 25, 30, 31). Our data indicate that strains of two other major lineages, CC1 and CC23, found in 79% of SKI and 62% of OAI, are frequently associated with skin, bone, and joint infections. The similar phylogenetic characteristics of strains isolated from skin and bone or joint infections provide support for the hypothesis that the skin and soft tissues may be a potential portal of entry for bone and joint infections, as previously suggested (14).
The high percentages of CC1 and CC23 strains observed in SKI and OAI suggested that strains of these lineages have enhanced invasiveness for skin, bone, and joints. As for the propensity of ST-17 strains that invade the CNS of neonates, the pathogenic features of the strains of the two phylogenetically distant lineages CC1 and CC23, which may account for the particular ability of these strains to invade skin, bone, and joints, are unknown. In terms of evolution, the strains of these two lineages probably acquired virulence through the acquisition of different genetic elements. Indeed, the strains of these two lineages displayed a high degree of phylogenetic divergence (6, 20, 21, 24, 31, 32, 43, 48). In addition, the CC23 lineage contains strains of bovine origin isolated during the 1960s and strains subsequently isolated from humans (7, 21). In contrast, the CC1 lineage contains strains that have emerged since the 1990s, responsible for infections in both adults and neonates (6, 17, 32). Thus, the bacteria of these two clones have been exposed to different environmental and nutritional backgrounds during evolution. These constraints may have subjected the bacteria to different stressful conditions, resulting in the induction of different mutations, as observed for housekeeping genes, and probably also resulting in differences in horizontal gene transfer events, leading to marked differences in the virulence proprieties of the strains of these two clones.
Our data are consistent with the hypothesis that horizontal gene transfers related to transduction mechanisms may have played a major role in the emergence of clones able to infect skin, bone, and joints. Indeed, on the basis of the prophage content of the S. agalactiae strain genomes, we identified two major groups of strains responsible for SKI and OAI with (i) very distinctive prophage DNA fragment contents, resulting in clustering into two particular prophage DNA groups, and (ii) the largest number of prophage DNA fragments per strain in their genome (groups B and D) (Fig. (Fig.22).
Prophage DNA group D had the largest number of amplified prophage DNA fragments per strain and contained all the strains of ST-1 and 85% of the CC1 strains, this phylogenetic lineage being the most frequently implicated in SKI and OAI. In addition, CC1 strains from prophage DNA groups other than group D were rarely isolated from SKI or OAI cases, with only six such isolations observed. These strains belonged to prophage DNA groups from which only one or two prophage DNA fragments were amplified, consistent with a low prophage content in the genome. Therefore, lysogeny, which has been shown to play an important role in bacterial virulence and genome diversification in the genus Streptococcus (1, 8), may be a key genetic event affecting the virulence of CC1 S. agalactiae strains, leading to the emergence of strains particularly able to infect skin, bone, and joints.
CC23 was the second most frequently implicated lineage in SKI and OAI. Analysis of the prophage content of the genome of CC23 strains resulted in the grouping together of half these strains (14/30 strains) in prophage DNA group B, one of the prophage DNA groups with the largest number of similar amplified prophage DNA fragments, suggesting a role for lysogeny in the specialization of this group of strains. Within this prophage DNA group, the CC23 strains clustered with 64% of the CC19 strains (7/11 strains). These two groups of strains may therefore have been subjected to similar ecological conditions or similar constraints leading to lysogenization by similar phages. However, the CC23 strains of prophage DNA group B were frequently associated with SKI and OAI (10/14 strains), whereas the CC19 strains of this prophage DNA group were rarely isolated from patients with these diseases (2/7 strains). Thus, the prophage characteristics of prophage DNA group B recognized by PCR in this study either play no role in the propensity of CC23 strains to infect skin, bone, and joints or may modulate other virulence factors specifically carried by the genomes of the strains of the CC23 lineage. Our data tend to support the second hypothesis. Indeed, CC23 strains from prophage DNA groups B and D, which had the greatest amplified prophage DNA content, were frequently isolated from SKI and OAI (14/18 strains), whereas CC23 strains from other prophage DNA groups, in which prophage DNA fragment amplification was less frequent, were mostly isolated from cases of HCol (8/12 strains; P = 0.014).
In conclusion, our data suggest a role for S. agalactiae strains of two phylogenetic lineages, CC1 and CC23, in SKI and OAI in adults, particularly for strains exposed to particular transduction mechanisms in gene recombination. The impacts of lysogeny on the virulence of the strains of these two lineages may be markedly different. Further studies are therefore required to assess in detail the role of the observed prophage-like elements in the emergence of S. agalactiae clones displaying a particular tropism for skin, bone, or joints. Are these elements involved in the importation of new phage-encoded virulence factors or the modification of transcriptional control mechanisms for chromosomal virulence genes? Particular host signals, linked to risk factors for development of SKI or OAI, may also induce changes in gene expression due to phage transduction.
N.V.D.M.-M. and R.Q. conceived and designed the experiments. M.S. (serotyping, MLST, prophage PCR, and PFGE) and A.-S.D. and L.A. (prophage PCR) performed the experiments. M.S., N.V.D.M.-M, and R.Q. analyzed the data. M.S. and R.Q. wrote the paper.
Published ahead of print on 24 February 2010.