G Model
ARTICLE IN PRESS
IJMM 50826 1–8
International Journal of Medical Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm
Phenotypic and molecular characterization of hyperpigmented group B Streptococci
1
2
3 4 5 6 7 8
Q1
Agnese Lupo a , Corinne Ruppen a , Andrew Hemphill b , Barbara Spellerberg c , Parham Sendi a,d,∗ a
Institute for Infectious Diseases, University of Bern, Bern, Switzerland Institute of Parasitology, University of Bern, Bern, Switzerland c Institute of Medical Microbiology and Hygiene, University Hospital Ulm, Ulm, Germany d Department of Infectious Diseases, Bern University Hospital, Bern, Switzerland b
9
10 23
a r t i c l e
i n f o
a b s t r a c t
11 12 13 14 15
Article history: Received 12 November 2013 Received in revised form 5 May 2014 Accepted 11 May 2014
16
22
Keywords: Group B streptococci Granadaene pigment cyl operon covS/R -Hemolysin
24
Introduction
17 18 19 20 21
25 26 27 28 29 30 31 32
Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. -hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that – at least in some clinical isolates – for -hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved. © 2014 Published by Elsevier GmbH.
Group B Streptococcus (GBS) is a major cause of sepsis in neonates and pregnant women. The incidence of invasive GBS disease in nonpregnant adults is growing, in particular in elderly persons and in those with chronic underlying conditions (e.g. diabetes mellitus). Cases of severe, life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome due to GBS have also been reported (Sendi et al., 2008). The development of GBS disease is based on bacterial colonization (e.g. of the vaginal epithelium),
∗ Corresponding author at: Department of Infectious Diseases, Bern University Hospital, Bern, Switzerland. Tel.: +41 31 632 99 99; fax: +41 31 632 87 66. E-mail address: parham.sendi@ifik.unibe.ch (P. Sendi).
penetration of epithelial barriers and invasion into sterile compartments. GBS expresses a diverse array of virulence factors that mediate specific host–cell interactions and interfere with innate immune clearance mechanisms (Liu and Nizet, 2004; Maisey et al., 2008). The surface-associated toxin -hemolysin/cytolysin (-h/c) is a crucial GBS virulence factor. Production of -h/c is associated with direct lysis of a variety of eukaryotic cell types (Gibson et al., 1999), inflammatory activation (Doran et al., 2002) and virulence in animal models (Ring et al., 2002). GBS isolates can be hyperpigmented when they express an orange-reddish pigment, a phenomenon that has also been linked to -h/c expression. Hyperpigmented GBS have been reported to be hyperhemolytic (Nizet et al., 1996). Hence, in invasive GBS diseases, a significant pathogenetic role has been attributed to the pigment (Liu et al., 2004;
http://dx.doi.org/10.1016/j.ijmm.2014.05.003 1438-4221/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
G Model IJMM 50826 1–8
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
2
81
Sendi et al., 2009; Whidbey et al., 2013). -h/c is encoded by the genes of the cyl operon (Spellerberg et al., 1999; Pritzlaff et al., 2001). Biosynthesis of the pigment also requires the 12-gene cyl operon (Spellerberg et al., 1999; Liu et al., 2004), with the link to pigment expression having recently been proposed to lie on several genes within this operon. The genes encode enzymes catalyzing different steps in fatty acid biosynthesis (Whidbey et al., 2013). Targeted mutations of different cyl genes result in a nonhemolytic and non-hyperpigmented GBS phenotype (Spellerberg et al., 1999; Liu et al., 2004; Gottschalk et al., 2006). We previously discovered two phenotypically different (i.e. hyperpigmented and non-hyperpigmented) but clonally identical GBS isolates that showed no mutation in the cylE and cylA genes. In the hyperpigmented variant, the covR sequence had a three-base-pair deletion (Sendi et al., 2009). The two-component regulatory system CovS/R (Control of virulence S/R) consists of CovS (sensor), which acts as a kinase, and CovR (regulator), which activates the response. A complete deletion of covS/R results in up-regulation of cyl genes and downregulation of genes in the cps operon for capsule production. Additionally, covS/R mutants show reduced CAMP activity (Lamy et al., 2004; Jiang et al., 2005). Interestingly, while previous studies on the regulation and virulence of the GBS pigment were performed on laboratory strains (i.e. isogenic mutants) (Lamy et al., 2004; Liu et al., 2004; Jiang et al., 2005; Whidbey et al., 2013), our findings were discovered in a clinical isolate obtained from a patient with necrotizing fasciitis and toxic shock syndrome (Sendi et al., 2009). To further elucidate these findings in clinical isolates, we prospectively collected hyperpigmented GBS isolates that were identified in a routine diagnostic microbiology laboratory. We then performed phenotypic and genetic analyses of the isolates. We thereby aimed to identify the spectrum of variations within the CovS/R system in clinical hyperpigmented GBS isolates.
82
Materials and methods
83
Bacterial isolates
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
105 106
107 108
GBS species was determined in all isolates by selective agar media, agglutination test and matrix-assisted laser desorption/ ionization/time-of-flight mass spectrometry. Hyperpigmented GBS isolates were looked for and prospectively collected from July 2008 until December 2012 during routine diagnostic procedures in the microbiology laboratory of the Institute of Infectious Diseases, University of Bern, Switzerland. To delineate hyperpigmented isolates from non-hyperpigmented isolates, we required four criteria: (1) reddish pigmentation in the center of a colony on a Columbia blood agar plate; (2) reddish pellet of a Todd Hewitt Broth (THB) culture after centrifuging and washing with phosphate buffer solution (PBS); (3) a reddish pellet after washing with dimethyl sulfoxide (DMSO); and (4) a typical absorbance spectrum with peaks at 455, 485 and 520 nm (Merritt and Jacobs, 1978; Tapsall, 1986; Liu et al., 2004). Because we aimed to unambiguously support the delineation of hyperpigmented from non-hyperpigmented isolates, pigments from 22 randomly selected GBS isolates from a routine diagnostic laboratory were purified and subjected to spectrophotometric analysis. If a hyperpigmented isolate was selected, we looked for phenotypic variants in the same specimen. We used NEM316 and ATC13813 as control strains. Antimicrobial susceptibility, serotype determination, clonality analyses The minimum inhibitory concentrations of penicillin, clindamycin and erythromycin were determined twice with Etests
(bioMérieux, Marcy l’Etoile, France), performed and interpreted as recommended by the Clinical and Laboratory Standards Institute for broth microdilution (CLSI, 2010). Serotyping was performed by using a rapid latex agglutination test (Strep-B-Latex kit, Statens Serum Institut, Copenhagen, Denmark) (Slotved et al., 2003) and confirmed by multiplex PCR (Imperi et al., 2010). The clonal complex of the isolates was determined by multilocus sequence typing (MLST), as described previously (Jones et al., 2003), using eBURSTv3 software (http://eburst.mlst.net/v3). If various GBS phenotypes were observed within the same specimen, clonal relatedness was evaluated. This was investigated by macrorestriction using SacII enzyme (Promega, Madison, WI, USA). Fragments were resolved by pulsed field gel electrophoresis (PFGE) for 21 h, with 1 s and 35 s as the initial and final impulse time, respectively, at 6 V/cm and 14 ◦ C. Analysis of pigment, hemolytic activity and capsule production Pigment was extracted from bacterial cultures as described previously (Rosa-Fraile et al., 2006; Whidbey et al., 2013). The absorbance of the purified pigment was measured with a spectrophotometer (Cary 300 Bio, UV–vis, Varian, USA) from 410 to 610 nm. To estimate the absorbance intensity of the pigment in selected hyperpigmented isolates in comparison to that of NEM316, we added a mathematically calculated borderline to each spectra graph. This borderline was defined to be more than at least one logarithmic unit above the absorbance spectra of NEM316 at every measurement point. Hemolytic activity was determined by performing 10-fold serial dilutions of the pigment extract in PBS containing 0.2% glucose and incubating with 1% sheep blood at 37 ◦ C for 1 h (Whidbey et al., 2013). The release of hemoglobin was estimated by optical density at 420 nm. The values obtained at a dilution of 10−4 were compared with those obtained from 1% sheep blood treated with a 0.1% sodium dodecyl sulfate solution. To evaluate the association between the absorbance intensity of the pigment and hemolytic activity, we correlated data on pigment absorption peaks at 455, 485 and 520 nm with results from the hemolytic activity assay. The presence of the capsule was determined by using the following two methods: 1. Buoyance density gradient stratification (Buchanan et al., 2005). Bacterial cultures were harvested by centrifugation and the pellets washed with PBS, followed by centrifugation. The pellets were resuspended in 1 ml of PBS and loaded on a centrifugation gradient obtained by stratification of Ficoll 400 (Pharmacia) solutions at 70, 60 and 50%. 2. Transmission electron microscopy (TEM). Bacterial isolates were incubated in THB to an OD of 0.8 (absorbance 600 nm). Isolates were then washed and fixed with Karnovsky solution. After polymerization, samples were sectioned with an ultramicrotome (Ultracut E ultramicrotome, Reichert-Jung, Vienna, Austria) before TEM imaging (Phillips EM 400 operating at 60 kV). Molecular analysis Genomic DNA was obtained according to the procedure of Murray et al. (1990). Fifty nanograms was used in the PCR mixture reactions. The amplifications were performed with Q5 High-Fidelity Master Mix (New England BioLab, Ipswich, MA, USA). Denaturation and amplification steps were performed at the conditions suggested by the manufacturer. All primers used in the study and melting temperatures are listed in Table S1 (supplementary files). Amplified targets for covS/R and part of the cyl operon were
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147
148 149 150 151 152 153 154 155 156 157 158 159 160
161
162 163 164 165 166 167 168 169
G Model IJMM 50826 1–8
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
170 171 172 173 174 175 176 177 178
179
180 181 182 183 184 185 186 187 188 189 190 191 192 193 194
195
196 197 198
sequenced, and results were analyzed with the NCBI nucleotide Blast program (http://blast.ncbi.nlm.nih.gov). The sequences relative to covS/R were compared with those deposited in GenBank with accession number AL766852.1 by using ClustalW. In addition, we sequenced the CovR binding site to the cyl operon, which is internal to fragment 660346-660364 of nucleotide sequence accession number NC 004368.1. Supplementary Table related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003. RNA isolation and quantification RNA was extracted from cultures in THB at absorbance 0.8 (600 nm) as previously described (Sendi et al., 2009). Contaminant DNA was eliminated by treating extracts with the TURBO DNA free kit (Applied Biosystem Inc., Foster City, CA, USA). To confirm the absence of DNA, we used the RNA extracts as template in a PCR reaction. Retro-transcription and real-time quantitative PCR (RTqPCR) were performed using the GoTaq 2-Step RT-qPCR System (Promega, USA). Expression of selected virulence genes (cfb, cpsG and cylE) in strains harboring a covS/R modification was evaluated by absolute quantification of the mRNA transcripts normalized to the expression level of the gyrA housekeeping gene. For this aim, we performed a calibration curve for each gene as previously described (Florindo et al., 2012). In addition, transcriptional analyses between clonally identical but phenotypically different variants were conducted with the 2−Ct method (Livak and Schmittgen, 2001). Statistical analysis GraphPad Prism 5.0 was used for building graphs and for statistical analysis (Pearson r and Student’s t-test where appropriate). A two-tailed p value of ≤0.05 was considered significant.
199
Results
200
Bacterial isolates
3
Pigment detection and hemolytic activity Photos of the colonies, the DMSO-washed pellets and the absorbance spectra of the GBS isolates are displayed in Fig. 1. The NEM316 pigment showed a typical spectrum with peaks at 455, 485 and 520 nm, whereas neither ATCC13813 nor one of the 22 randomly selected isolates showed absorbance at these wavelengths (supplementary file, Fig. S2A and S2C). The mathematically calculated borderline (i.e. reflecting data that are at least one logarithmic unit above the absorbance spectra of NEM316 at every measurement point) was set at 0.15 (supplementary file, Fig. S2B). The absorbance ranges of hyperpigmented isolates were 0.17–1.61, 0.23–2.19 and 0.17–1.91 at 455, 485 and 525 nm, respectively. The results of the hemolytic activity assays are represented in Fig. 2A. We observed a significant correlation between hemolytic activity (Fig. 2B) and pigment absorbance values obtained at 455 nm (Pearson r = 0.81, 95% CI 0.46–0.94, p = 0.0008), 485 nm (Pearson r = 0.80, 95% CI 0.43–0.94, p = 0.0012) and 520 nm (Pearson r = 0.79, 95% CI 0.41–0.93, p = 0.0015). Supplementary Figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003. Molecular analysis of the hyperpigmented GBS isolates Molecular alterations within the CovS/R sites are presented in Fig. 3A. The alterations included a three-base-pair deletion (Val31) (isolate 001) and two nucleotide substitutions leading to Ala90Asp (isolate 013) and Arg119His (isolate 016), respectively. Further, in isolate 014 (both variants A and B), there was a nucleotide insertion leading to a truncated CovR at amino acid 206. In variant B of isolate 010, there was a 13-nucleotide deletion causing an abortive CovR (stop at amino acid 11) and an IS1381 inserted between the ribosome-binding site and the CovR binding site upstream of the covR gene. In isolate 012, the analysis of the promotor region of the cyl operon revealed a 19-base deletion from −295 to −277, from the cylX start codon, overlapping the CovR binding site (−281 to −252) (Fig. 3B). No covS/R variations were found in hyperpigmented isolates 002, 003, 008 and 015B. Transcriptional analysis and phenotypic tests of virulence factors
201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226
We identified 10 GBS hyperpigmented isolates obtained from 10 individuals. In seven patients, there was only one phenotype. In three patients, the hyperpigmented isolate was accompanied by a phenotypically different variant (i.e. variant A and B). In two of them (010, 015), one variant was considered non-hyperpigmented (variant A), whereas in one isolate (014) both variants showed an orange-reddish pigment. However, in one variant of 014 (variant B), the pigment was more reddish and the absorbance more intense (Fig. 1). PFGE analysis suggested that variants of the three isolates (010, 014, 015) were indistinguishable (supplementary file, Fig. S1). Hence, 13 isolates in total were included in this study. The source and characterization of the isolates is presented in Table 1. Three isolates were obtained from patients who had no disease manifestation (i.e. colonization) and seven isolates from sites in patients with a clinical syndrome. Disease manifestation varied from nonsevere (conjunctivitis, tonsillopharyngitis) to very severe (necrotizing fasciitis). None of the capsular serotypes was significantly associated with hyperpigmentation. The results of MSLT analysis are presented in Table 1. Three isolates were single locus variants of other sequence types (i.e. isolate 008 ST335 = variant of ST19, isolates 010A/B ST255 = variants of ST6, isolate 016 ST9 = variant of ST10). Apart from isolate 015A/B (singleton ST130), 10 isolates belonged to clonal complex (CC) 19, and one to CC17. All isolates were susceptible to penicillin, erythromycin and clindamycin. Supplementary Figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003.
We then quantified the transcripts of selected virulence genes in isolates harboring covS/R modifications and their variants (isolates 001, 010A and B, 013, 014A and B, 016) as well as that of NEM316 (control). Transcription of the cpsG gene was low in all isolates. The cylE gene was overexpressed in all isolates, except in variant A of isolate 010, which had a functional CovR. It was also significantly overexpressed when compared with cfb and cpsG in all isolates but 010A and 014A (Fig. 4). The expression patterns of 010A and its hyperpigmented variant 010B, which has a truncated CovR, were in agreement with the observed phenotypes of the isolates (Fig. 4 and supplementary file, Fig. S3), confirming the role of the CovS/R system as observed previously (Sendi et al., 2009). Variant B of isolate 014, which harbored the same CovR mutation as that of 014A, showed an overexpression of the cylE gene compared with the cfb and cpsG genes. Surprisingly, the cfb gene was overexpressed in variant 014B (Fig. 4). These analyzed transcript levels were in agreement with phenotypic profiles (supplementary file, Fig. S3). Supplementary Figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003. Discussion In this study, we report the molecular and phenotypic characterization of a collection of clinical GBS isolates showing pronounced
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
227
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246
247
248 249 250 251 252 253 254 255 256 257 258 259 260 261
262
263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282
283
284 285
G Model IJMM 50826 1–8 4
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
Fig. 1. Photos of the colonies, the DMSO-washed pellets and the absorbance spectra of the GBS isolates.
286 287 288 289 290 291 292 293 294 295 296 297 298 299
pigmentation. To the best of our knowledge, the term hyperpigmentation is not precisely defined. The selection criteria applied in this study were based on phenotypic characteristics and analyses of pigment absorbance spectra. Each so-called hyperpigmented isolate included in this study had a pigment absorbance that was at least more than one log higher than those obtained from NEM316 (Fig. 1). Although more in-depth (e.g. chemical and physical) analysis is needed to define ‘hyperpigmented’ isolates, we believe that our approach appears reasonable and practicable for other research laboratories. The hyperpigmented isolates were collected from different anatomical sites and various patients. They were found as colonizers of the urogenital tract; in nonsevere diseases, such as tonsillopharyngitis and conjunctivitis; and in life-threatening
infections, such as necrotizing fasciitis. Thus, hyperpigmentation in clinical isolates is not uniformly associated with severe invasive disease. Recent advances in the understanding of the link between pigment production and -h/c activity in GBS have been achieved. Whidbey et al. (Whidbey et al., 2013) showed that the pigment consists of an ornithine rhamnolipid compound, and that this pigment has hemolytic and cytolytic activity. Consistent with these and previous results (Nizet et al., 1996), we found an overall good correlation between pigmentation and hemolytic activity (Fig. 2B). This was in particular obvious with isolates that revealed high absorbance values (i.e. 001, 008, 012, 010B and 014B). However, because we did not quantify the amount of the ornithine rhamnolipid compound, a direct correlation between pigment and
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
300 301 302 303 304 305 306 307 308 309 310 311 312 313
G Model
ARTICLE IN PRESS
IJMM 50826 1–8
A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
5
Table 1 Characterization of selected clinical GBS isolates. Isolate
Specimen
Patient
Clinical manifestation
Age
Sex
Biopsy Cervix swab Hip joint fluid Cervix swab Eye swab
74 22 88 20 1 month
M F M F M
Prosthetic joint infection No disease, colonization Septic arthritis No disease, colonization Conjunctivitis
Urine Biopsy Vaginal swab
64 77 55
F M F
Urine
65
M
Urinary tract infectionb Necrotizing fasciitis Bleeding from uterus myomatosus No disease, colonizationc
Throat swab
4
M
Tonsillopharnyngitis
Serotype
ST
MIC (E-test) P S ≤ 0.25
001 002 003 008 010Aa 010Ba 012 013 014Aa 014Ba 015Aa 015Ba 016
V III Ib III Ib Ib III III II II IX IX Ib
12 19 8 335 255 255 28 17 19 19 130 130 9
E R > 0.25
0.094 0.064 0.125 0.047 0.064 0.064 0.047 0.064 0.023 0.023 0.023 0.023 0.064
S ≤ 0.25
CLI R > 0.5
0.064 0.064 0.032 0.064 0.047 0.047 0.047 0.032 0.016 0.016 0.032 0.032 0.032
S ≤ 0.5
R > 0.5 0.094 0.094 0.094 0.094 0.25 0.25 0.064 0.064 0.047 0.047 0.125 0.125 0.094
M, male; F, female; ST, sequence type determined by MLST; MIC, minimal inhibitory concentration determined by E test; P, penicillin; E, erythromycin; CLI, clindamycin. a A and B correspond to clonally identical but phenotypically different variants: A, variant; B, hyperpigmented. b Polymicrobial infection. c Urine after bladder reconstruction and radiation after cancer.
314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333
hemolytic activity cannot be made. In addition, we found reddish colonies (e.g. 003, 015B) that did not reveal high absorbance values of the pigment extract or hemolytic activity. These discrepancies may be explained by variations in the chromophore part of the molecule, which would not affect its hemolytic property. Thus, pigment and hemolytic activity are often coupled, but this phenomenon cannot be applied to all clinical isolates. Moreover, results suggesting uniform correlation between pigment and hemolytic activity should be interpreted with caution because -h/c is an unstable cell-wall-associated toxin, and pure extraction of both pigment and -h/c is technically challenging. However, our study is the first to characterize pigmentation and hemolytic activity in a clinical collection of isolates with different genetic backgrounds and physiological features. In three specimens, hyperpigmented isolates (010B, 014B and 015B) were found together with an accompanying variant. Interestingly, in isolate 014, both variants were considered ‘hyperpigmented’, though they showed different levels of pigmentation. This phenomenon of clonally identical but phenotypically different GBS variants has been described previously in isolates obtained
from patients with severe disease (Sigge et al., 2008; Sendi et al., 2009). The hyperpigmented variants showed decreased CAMP and capsule production and carried mutations in the regulatory gene covR. However, in this study, the previously described characteristics were found in only one (010A and B) of the three paired variants. While the capsule was less expressed in all three hyperpigmented variants, the CAMP reaction was not decreased in isolates 014B and 015B (supplementary file, Fig. S3). Thus, although phenotypic characterization was performed in only three paired variants, it appears that in hyperpigmented isolates, capsule production is decreased. In contrast, an association between hyperpigmentation and a decrease in the CAMP reaction was observed in only one of the three isolates. We investigated a possible role of the covS/R system in hyperpigmented isolates and found that in five isolates, the covR sequence was mutated. These mutations caused truncation of CovR in two isolates (variant B of isolate 010 and both variants [A and B] of isolate 014) and a single amino acid deletion and substitution in the remaining three isolates (Val31, Ala90Asp and Arg119His in isolates 001, 013 and 016, respectively) (Fig. 3A). In addition, a partial
Fig. 2. (a) Hemolytic activity of GBS isolates. The values obtained at a dilution of 10−4 were compared with those obtained from 1% sheep blood treated with a 0.1% sodium dodecyl sulphate solution. (b) Association between absorbance intensity of the pigment at 455, 485 and 520 nm and hemolytic activity.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353
G Model IJMM 50826 1–8 6
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
Fig. 3. (a) Alignment of the CovR amino acid sequences from the isolates harboring mutations and highlights of the different residues compared with sequence NEM316 (accession number AL766852.1) (ClustalW). (b) Alignment of the cylX promoter region of isolate 012 and NEM316 (accession number AL766846.1). The CovR binding region (−281, −252 from ATG starting codon) proposed by Lamy et al. (2004) (Clustal W) is in bold and underlined.
354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
deletion in the CovR binding site (Pcyl region) in isolate 012 was detected (Fig. 3B). All of these mutations, except Val31 in isolate 001 (Sendi et al., 2009), are here reported for the first time in GBS. The functional consequences of the amino acid substitutions found in our study are unknown. They may be in line with the in vitro findings described by Lin et al. (2009) in GBS and Horstmann et al. (2011) in Group A Streptococcus (GAS). Promoter binding of CovR required phosphorylation of the aspartate residue at position 53. Phosphorylation of threonine at position 65 prevented CovR from binding to DNA (Lin et al., 2009). Expression of -h/c may thereby be de-repressed. Considering the homology and the similar functions between GAS and GBS CovR, a parallelism between the two structures can be speculated. The substitutions observed in the hyperpigmented isolates most likely affect the receiver domain of the CovR protein, probably interfering with the efficiency of phosphorylation by CovS. In particular, Arg119 substitution has been repeatedly described in GAS isolates (Horstmann et al., 2011). To confirm a role for the detected mutations in the regulatory pattern of our hyperpigmented isolates, we performed a
transcription analysis of the cfb (CAMP), cpsG (capsule) and cylE genes (hyperpigmentation). In hyperpigmented isolates (001, variant B of 010, 013, variants A and B of 014, and 016), CovR mutations were associated with the overexpression of the cylE gene and the downregulation of the cpsG gene. The findings in isolate 014 are consistent with the phenotypic observation that both variants showed an orange-reddish pigment. The downregulation of the cfb gene was not observed in variant B of isolate 014 or in isolate 016 (supplementary file, Fig. S3). The cfb gene was upregulated in variant A of isolate 010. Thus, in clinical hyperpigmented isolates with a mutated CovR, transcript analysis was uniformly consistent with cylE overexpression and cpsG downregulation, but downregulation of cfb (CAMP) was not found in all isolates. This parallels the abovedescribed phenotypic observations in the three isolates with paired variants (010, 014, 015). Considering that -h/c and the capsule are major virulence components, this investigation on clinical isolates supports the role of the CovS/R system in the regulation of these factors involved in GBS pathogenesis. Of note, CAMP appears not to be essential for systemic virulence in GBS (Hensler et al., 2008).
Fig. 4. cfb, cpsG and cylE gene expression was analyzed in the isolates harboring mutations in the covS/R sequence. For each isolate, we performed an absolute quantification of the mRNA transcripts normalized to the expression level of the gyrA housekeeping gene. The level of expression of the cylE gene was compared with that of the cfb and the cpsG genes by t-test.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
G Model IJMM 50826 1–8
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444
445
446 447 448 449 450 451 452 453
Our study has limitations. Hyperpigmented isolates were looked for on the basis of phenotypic characteristics, but in contrast to accepted characteristics of phenotypic variants in other species (e.g. small colony variants of Staphylococcus aureus (Sendi and Proctor, 2009)), there are no uniformly accepted characteristics to discriminate these GBS isolates. We focused on reddish isolates, and (photo) documented applied criteria that appeared reasonable to us. We also used several criteria because one criterion would be unreliable and no correlation can be made on visual parameters (e.g. phenotype on an agar plate versus product after DMSO treatment). However, these criteria may be unpractical for screening in a routine diagnostic laboratory. Selecting colonies for further investigation on the basis of phenotype through identification by laboratory staff may be an insensitive method. Also special media may be required (e.g. Columbia blood agar containing proteose peptone 3 and starch). Thus, it is possible that an unknown number of hyperpigmented isolates were missed during the observation period. Consequently, no firm incidence rate on hyperpigmented isolates can be given. The selection of 10 isolates during a 4.5-year period was found in an institute that registers approximately 350–400 GBS isolates per year. Yet, we have presented the largest published series of clinical GBS showing hyperpigmentation. Another limitation includes the fact that the array of genes co-regulated by CovR is wider than the number of genes analyzed in this study. On the other hand, the core CovS/R regulon comprises the genes selected in this study (Jiang et al., 2005). Finally, hyperpigmentation may be associated with mutations outside of the CovS/R pathways, since we found four isolates without mutations in the covS/R genes and their binding sites. Analyzing all possible genes potentially responsible for hyperpigmented phenotypes is, however, beyond the scope of this study. In conclusion, clinical hyperpigmented isolates are associated with increased hemolytic activity in vitro, although hyperpigmentation in GBS isolates is not strictly related to invasive diseases and mutations within the covS/R genes. In three of four isolates without genetic alterations, no associated clinical syndrome was present. In 6 of 10 isolates, mutations were found, but only two of them were obtained from patients with acute severe infections (periprosthetic joint infection and necrotizing fasciitis). The alterations found within the CovS/R system of hyperpigmented isolates were consistently correlated to increased cylE and decreased cpsG (capsule) expression. These findings can suggest a correlation between the alteration of the CovS/R system and the development of invasive disease, as previously postulated for isogenic mutants (Lamy et al., 2004; Liu et al., 2004; Jiang et al., 2005; Whidbey et al., 2013). However, covR mutations were also found in patients without clinical syndromes. Adding to the puzzle are the unpredicted results in the regulatory pattern of the analyzed genes (e.g. cfb [CAMP]) compared with in vitro investigations of isogenic mutants. This highlights the necessity of performing additional clinical studies to further elucidate the complex GBS regulatory network and its pathogenesis.
Acknowledgments This study was presented in part at the 7th International Conference on Gram-Positive Microorganisms, Montecatini Terme, Tuscany, Italy (P165). This work is supported by the Velux Stiftung, Zurich, Switzerland. We thank Drs. Andrea Endimiani and Sara Droz, and Gabriela Reichen, for critical discussion and technical support. The manuscript was edited by Barbara Every, BioMedical Editor, St. Albert, Alberta, Canada.
7
References Buchanan, J.T., Stannard, J.A., Lauth, X., Ostland, V.E., Powell, H.C., Westerman, M.E., Nizet, V., 2005. Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development. Infection and Immunity 73, 6935–6944. CLSI, 2010. Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing Twentieth Informational Supplement M100-S20. Clinical and Laboratory Standards Institute, Wayne, PA. Doran, K.S., Chang, J.C., Benoit, V.M., Eckmann, L., Nizet, V., 2002. Group B streptococcal beta-hemolysin/cytolysin promotes invasion of human lung epithelial cells and the release of interleukin-8. Journal of Infectious Diseases 185, 196–203. Florindo, C., Ferreira, R., Borges, V., Spellerberg, B., Gomes, J.P., Borrego, M.J., 2012. Selection of reference genes for real-time expression studies in Streptococcus agalactiae. Journal of Microbiological Methods 90, 220–227. Gibson, R.L., Nizet, V., Rubens, C.E., 1999. Group B streptococcal beta-hemolysin promotes injury of lung microvascular endothelial cells. Pediatric Research 45, 626–634. Gottschalk, B., Broker, G., Kuhn, M., Aymanns, S., Gleich-Theurer, U., Spellerberg, B., 2006. Transport of multidrug resistance substrates by the Streptococcus agalactiae hemolysin transporter. Journal of Bacteriology 188, 5984–5992. Hensler, M.E., Quach, D., Hsieh, C.J., Doran, K.S., Nizet, V., 2008. CAMP factor is not essential for systemic virulence of Group B Streptococcus. Microbial Pathogenesis 44, 84–88. Horstmann, N., Sahasrabhojane, P., Suber, B., Kumaraswami, M., Olsen, R.J., Flores, A., Musser, J.M., Brennan, R.G., Shelburne 3rd, S.A., 2011. Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis. PLoS Pathogens 7, e1002311. Imperi, M., Pataracchia, M., Alfarone, G., Baldassarri, L., Orefici, G., Creti, R., 2010. A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae. Journal of Microbiological Methods 80, 212–214. Jiang, S.M., Cieslewicz, M.J., Kasper, D.L., Wessels, M.R., 2005. Regulation of virulence by a two-component system in group B streptococcus. Journal of Bacteriology 187, 1105–1113. Jones, N., Bohnsack, J.F., Takahashi, S., Oliver, K.A., Chan, M.S., Kunst, F., Glaser, P., Rusniok, C., Crook, D.W., Harding, R.M., Bisharat, N., Spratt, B.G., 2003. Multilocus sequence typing system for group B Streptococcus. Journal of Clinical Microbiology 41, 2530–2536. Lamy, M.C., Zouine, M., Fert, J., Vergassola, M., Couve, E., Pellegrini, E., Glaser, P., Kunst, F., Msadek, T., Trieu-Cuot, P., Poyart, C., 2004. CovS/CovR of group B Streptococcus: a two-component global regulatory system involved in virulence. Molecular Microbiology 54, 1250–1268. Lin, W.J., Walthers, D., Connelly, J.E., Burnside, K., Jewell, K.A., Kenney, L.J., Rajagopal, L., 2009. Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR. Molecular Microbiology 71, 1477–1495. Liu, G.Y., Doran, K.S., Lawrence, T., Turkson, N., Puliti, M., Tissi, L., Nizet, V., 2004. Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense. Proceedings of the National Academy of Sciences of the United States of America 101, 14491–14496. Liu, G.Y., Nizet, V., 2004. Extracellular virulence factors of group B Streptococci. Frontiers in bioscience: A Journal and Virtual Library 9, 1794–1802. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Maisey, H.C., Doran, K.S., Nizet, V., 2008. Recent advances in understanding the molecular basis of group B Streptococcus virulence. Expert Reviews in Molecular Medicine 10, e27. Merritt, K., Jacobs, N.J., 1978. Characterization and incidence of pigment production by human clinical group B streptococci. Journal of Clinical Microbiology 8, 105–107. Murray, B.E., Singh, K.V., Heath, J.D., Sharma, B.R., Weinstock, G.M., 1990. Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology 28, 2059–2063. Nizet, V., Gibson, R.L., Chi, E.Y., Framson, P.E., Hulse, M., Rubens, C.E., 1996. Group B streptococcal beta-hemolysin expression is associated with injury of lung epithelial cells. Infection and Immunity 64, 3818–3826. Pritzlaff, C.A., Chang, J.C., Kuo, S.P., Tamura, G.S., Rubens, C.E., Nizet, V., 2001. Genetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Molecular Microbiology 39, 236–247. Ring, A., Braun, J.S., Pohl, J., Nizet, V., Stremmel, W., Shenep, J.L., 2002. Group B streptococcal beta-hemolysin induces mortality and liver injury in experimental sepsis. Journal of Infectious Diseases 185, 1745–1753. Rosa-Fraile, M., Rodriguez-Granger, J., Haidour-Benamin, A., Cuerva, J.M., Sampedro, A., 2006. Granadaene: proposed structure of the group B Streptococcus polyenic pigment. Applied and Environmental Microbiology 72, 6367–6370. Sendi, P., Johansson, L., Dahesh, S., Van-Sorge, N.M., Darenberg, J., Norgren, M., Sjolin, J., Nizet, V., Norrby-Teglund, A., 2009. Bacterial phenotype variants in group B streptococcal toxic shock syndrome. Emerging Infectious Diseases 15, 223–232. Sendi, P., Johansson, L., Norrby-Teglund, A., 2008. Invasive group B Streptococcal disease in non-pregnant adults: a review with emphasis on skin and soft-tissue infections. Infection 36, 100–111. Sendi, P., Proctor, R.A., 2009. Staphylococcus aureus as an intracellular pathogen: the role of small colony variants. Trends in Microbiology 17, 54–58.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
454
455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537
G Model IJMM 50826 1–8 8 538 539 540 541 542 543 544
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
Sigge, A., Schmid, M., Mauerer, S., Spellerberg, B., 2008. Heterogeneity of hemolysin expression during neonatal Streptococcus agalactiae sepsis. Journal of Clinical Microbiology 46, 807–809. Slotved, H.C., Elliott, J., Thompson, T., Konradsen, H.B., 2003. Latex assay for serotyping of group B Streptococcus isolates. Journal of Clinical Microbiology 41, 4445–4447. Spellerberg, B., Pohl, B., Haase, G., Martin, S., Weber-Heynemann, J., Lutticken, R., 1999. Identification of genetic determinants for the hemolytic activity of
Streptococcus agalactiae by ISS1 transposition. Journal of Bacteriology 181, 3212–3219. Tapsall, J.W., 1986. Pigment production by Lancefield-group-B streptococci (Streptococcus agalactiae). Journal of Medical Microbiology 21, 75–81. Whidbey, C., Harrell, M.I., Burnside, K., Ngo, L., Becraft, A.K., Iyer, L.M., Aravind, L., Hitti, J., Waldorf, K.M., Rajagopal, L., 2013. A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta. Journal of Experimental Medicine 210, 1265–1281.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
545 546 547 548 549 550 551 552
ARTICLE IN PRESS
IJMM 50826 1–8
International Journal of Medical Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm
Phenotypic and molecular characterization of hyperpigmented group B Streptococci
1
2
3 4 5 6 7 8
Q1
Agnese Lupo a , Corinne Ruppen a , Andrew Hemphill b , Barbara Spellerberg c , Parham Sendi a,d,∗ a
Institute for Infectious Diseases, University of Bern, Bern, Switzerland Institute of Parasitology, University of Bern, Bern, Switzerland c Institute of Medical Microbiology and Hygiene, University Hospital Ulm, Ulm, Germany d Department of Infectious Diseases, Bern University Hospital, Bern, Switzerland b
9
10 23
a r t i c l e
i n f o
a b s t r a c t
11 12 13 14 15
Article history: Received 12 November 2013 Received in revised form 5 May 2014 Accepted 11 May 2014
16
22
Keywords: Group B streptococci Granadaene pigment cyl operon covS/R -Hemolysin
24
Introduction
17 18 19 20 21
25 26 27 28 29 30 31 32
Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. -hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that – at least in some clinical isolates – for -hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved. © 2014 Published by Elsevier GmbH.
Group B Streptococcus (GBS) is a major cause of sepsis in neonates and pregnant women. The incidence of invasive GBS disease in nonpregnant adults is growing, in particular in elderly persons and in those with chronic underlying conditions (e.g. diabetes mellitus). Cases of severe, life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome due to GBS have also been reported (Sendi et al., 2008). The development of GBS disease is based on bacterial colonization (e.g. of the vaginal epithelium),
∗ Corresponding author at: Department of Infectious Diseases, Bern University Hospital, Bern, Switzerland. Tel.: +41 31 632 99 99; fax: +41 31 632 87 66. E-mail address: parham.sendi@ifik.unibe.ch (P. Sendi).
penetration of epithelial barriers and invasion into sterile compartments. GBS expresses a diverse array of virulence factors that mediate specific host–cell interactions and interfere with innate immune clearance mechanisms (Liu and Nizet, 2004; Maisey et al., 2008). The surface-associated toxin -hemolysin/cytolysin (-h/c) is a crucial GBS virulence factor. Production of -h/c is associated with direct lysis of a variety of eukaryotic cell types (Gibson et al., 1999), inflammatory activation (Doran et al., 2002) and virulence in animal models (Ring et al., 2002). GBS isolates can be hyperpigmented when they express an orange-reddish pigment, a phenomenon that has also been linked to -h/c expression. Hyperpigmented GBS have been reported to be hyperhemolytic (Nizet et al., 1996). Hence, in invasive GBS diseases, a significant pathogenetic role has been attributed to the pigment (Liu et al., 2004;
http://dx.doi.org/10.1016/j.ijmm.2014.05.003 1438-4221/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
G Model IJMM 50826 1–8
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
2
81
Sendi et al., 2009; Whidbey et al., 2013). -h/c is encoded by the genes of the cyl operon (Spellerberg et al., 1999; Pritzlaff et al., 2001). Biosynthesis of the pigment also requires the 12-gene cyl operon (Spellerberg et al., 1999; Liu et al., 2004), with the link to pigment expression having recently been proposed to lie on several genes within this operon. The genes encode enzymes catalyzing different steps in fatty acid biosynthesis (Whidbey et al., 2013). Targeted mutations of different cyl genes result in a nonhemolytic and non-hyperpigmented GBS phenotype (Spellerberg et al., 1999; Liu et al., 2004; Gottschalk et al., 2006). We previously discovered two phenotypically different (i.e. hyperpigmented and non-hyperpigmented) but clonally identical GBS isolates that showed no mutation in the cylE and cylA genes. In the hyperpigmented variant, the covR sequence had a three-base-pair deletion (Sendi et al., 2009). The two-component regulatory system CovS/R (Control of virulence S/R) consists of CovS (sensor), which acts as a kinase, and CovR (regulator), which activates the response. A complete deletion of covS/R results in up-regulation of cyl genes and downregulation of genes in the cps operon for capsule production. Additionally, covS/R mutants show reduced CAMP activity (Lamy et al., 2004; Jiang et al., 2005). Interestingly, while previous studies on the regulation and virulence of the GBS pigment were performed on laboratory strains (i.e. isogenic mutants) (Lamy et al., 2004; Liu et al., 2004; Jiang et al., 2005; Whidbey et al., 2013), our findings were discovered in a clinical isolate obtained from a patient with necrotizing fasciitis and toxic shock syndrome (Sendi et al., 2009). To further elucidate these findings in clinical isolates, we prospectively collected hyperpigmented GBS isolates that were identified in a routine diagnostic microbiology laboratory. We then performed phenotypic and genetic analyses of the isolates. We thereby aimed to identify the spectrum of variations within the CovS/R system in clinical hyperpigmented GBS isolates.
82
Materials and methods
83
Bacterial isolates
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
105 106
107 108
GBS species was determined in all isolates by selective agar media, agglutination test and matrix-assisted laser desorption/ ionization/time-of-flight mass spectrometry. Hyperpigmented GBS isolates were looked for and prospectively collected from July 2008 until December 2012 during routine diagnostic procedures in the microbiology laboratory of the Institute of Infectious Diseases, University of Bern, Switzerland. To delineate hyperpigmented isolates from non-hyperpigmented isolates, we required four criteria: (1) reddish pigmentation in the center of a colony on a Columbia blood agar plate; (2) reddish pellet of a Todd Hewitt Broth (THB) culture after centrifuging and washing with phosphate buffer solution (PBS); (3) a reddish pellet after washing with dimethyl sulfoxide (DMSO); and (4) a typical absorbance spectrum with peaks at 455, 485 and 520 nm (Merritt and Jacobs, 1978; Tapsall, 1986; Liu et al., 2004). Because we aimed to unambiguously support the delineation of hyperpigmented from non-hyperpigmented isolates, pigments from 22 randomly selected GBS isolates from a routine diagnostic laboratory were purified and subjected to spectrophotometric analysis. If a hyperpigmented isolate was selected, we looked for phenotypic variants in the same specimen. We used NEM316 and ATC13813 as control strains. Antimicrobial susceptibility, serotype determination, clonality analyses The minimum inhibitory concentrations of penicillin, clindamycin and erythromycin were determined twice with Etests
(bioMérieux, Marcy l’Etoile, France), performed and interpreted as recommended by the Clinical and Laboratory Standards Institute for broth microdilution (CLSI, 2010). Serotyping was performed by using a rapid latex agglutination test (Strep-B-Latex kit, Statens Serum Institut, Copenhagen, Denmark) (Slotved et al., 2003) and confirmed by multiplex PCR (Imperi et al., 2010). The clonal complex of the isolates was determined by multilocus sequence typing (MLST), as described previously (Jones et al., 2003), using eBURSTv3 software (http://eburst.mlst.net/v3). If various GBS phenotypes were observed within the same specimen, clonal relatedness was evaluated. This was investigated by macrorestriction using SacII enzyme (Promega, Madison, WI, USA). Fragments were resolved by pulsed field gel electrophoresis (PFGE) for 21 h, with 1 s and 35 s as the initial and final impulse time, respectively, at 6 V/cm and 14 ◦ C. Analysis of pigment, hemolytic activity and capsule production Pigment was extracted from bacterial cultures as described previously (Rosa-Fraile et al., 2006; Whidbey et al., 2013). The absorbance of the purified pigment was measured with a spectrophotometer (Cary 300 Bio, UV–vis, Varian, USA) from 410 to 610 nm. To estimate the absorbance intensity of the pigment in selected hyperpigmented isolates in comparison to that of NEM316, we added a mathematically calculated borderline to each spectra graph. This borderline was defined to be more than at least one logarithmic unit above the absorbance spectra of NEM316 at every measurement point. Hemolytic activity was determined by performing 10-fold serial dilutions of the pigment extract in PBS containing 0.2% glucose and incubating with 1% sheep blood at 37 ◦ C for 1 h (Whidbey et al., 2013). The release of hemoglobin was estimated by optical density at 420 nm. The values obtained at a dilution of 10−4 were compared with those obtained from 1% sheep blood treated with a 0.1% sodium dodecyl sulfate solution. To evaluate the association between the absorbance intensity of the pigment and hemolytic activity, we correlated data on pigment absorption peaks at 455, 485 and 520 nm with results from the hemolytic activity assay. The presence of the capsule was determined by using the following two methods: 1. Buoyance density gradient stratification (Buchanan et al., 2005). Bacterial cultures were harvested by centrifugation and the pellets washed with PBS, followed by centrifugation. The pellets were resuspended in 1 ml of PBS and loaded on a centrifugation gradient obtained by stratification of Ficoll 400 (Pharmacia) solutions at 70, 60 and 50%. 2. Transmission electron microscopy (TEM). Bacterial isolates were incubated in THB to an OD of 0.8 (absorbance 600 nm). Isolates were then washed and fixed with Karnovsky solution. After polymerization, samples were sectioned with an ultramicrotome (Ultracut E ultramicrotome, Reichert-Jung, Vienna, Austria) before TEM imaging (Phillips EM 400 operating at 60 kV). Molecular analysis Genomic DNA was obtained according to the procedure of Murray et al. (1990). Fifty nanograms was used in the PCR mixture reactions. The amplifications were performed with Q5 High-Fidelity Master Mix (New England BioLab, Ipswich, MA, USA). Denaturation and amplification steps were performed at the conditions suggested by the manufacturer. All primers used in the study and melting temperatures are listed in Table S1 (supplementary files). Amplified targets for covS/R and part of the cyl operon were
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147
148 149 150 151 152 153 154 155 156 157 158 159 160
161
162 163 164 165 166 167 168 169
G Model IJMM 50826 1–8
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
170 171 172 173 174 175 176 177 178
179
180 181 182 183 184 185 186 187 188 189 190 191 192 193 194
195
196 197 198
sequenced, and results were analyzed with the NCBI nucleotide Blast program (http://blast.ncbi.nlm.nih.gov). The sequences relative to covS/R were compared with those deposited in GenBank with accession number AL766852.1 by using ClustalW. In addition, we sequenced the CovR binding site to the cyl operon, which is internal to fragment 660346-660364 of nucleotide sequence accession number NC 004368.1. Supplementary Table related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003. RNA isolation and quantification RNA was extracted from cultures in THB at absorbance 0.8 (600 nm) as previously described (Sendi et al., 2009). Contaminant DNA was eliminated by treating extracts with the TURBO DNA free kit (Applied Biosystem Inc., Foster City, CA, USA). To confirm the absence of DNA, we used the RNA extracts as template in a PCR reaction. Retro-transcription and real-time quantitative PCR (RTqPCR) were performed using the GoTaq 2-Step RT-qPCR System (Promega, USA). Expression of selected virulence genes (cfb, cpsG and cylE) in strains harboring a covS/R modification was evaluated by absolute quantification of the mRNA transcripts normalized to the expression level of the gyrA housekeeping gene. For this aim, we performed a calibration curve for each gene as previously described (Florindo et al., 2012). In addition, transcriptional analyses between clonally identical but phenotypically different variants were conducted with the 2−Ct method (Livak and Schmittgen, 2001). Statistical analysis GraphPad Prism 5.0 was used for building graphs and for statistical analysis (Pearson r and Student’s t-test where appropriate). A two-tailed p value of ≤0.05 was considered significant.
199
Results
200
Bacterial isolates
3
Pigment detection and hemolytic activity Photos of the colonies, the DMSO-washed pellets and the absorbance spectra of the GBS isolates are displayed in Fig. 1. The NEM316 pigment showed a typical spectrum with peaks at 455, 485 and 520 nm, whereas neither ATCC13813 nor one of the 22 randomly selected isolates showed absorbance at these wavelengths (supplementary file, Fig. S2A and S2C). The mathematically calculated borderline (i.e. reflecting data that are at least one logarithmic unit above the absorbance spectra of NEM316 at every measurement point) was set at 0.15 (supplementary file, Fig. S2B). The absorbance ranges of hyperpigmented isolates were 0.17–1.61, 0.23–2.19 and 0.17–1.91 at 455, 485 and 525 nm, respectively. The results of the hemolytic activity assays are represented in Fig. 2A. We observed a significant correlation between hemolytic activity (Fig. 2B) and pigment absorbance values obtained at 455 nm (Pearson r = 0.81, 95% CI 0.46–0.94, p = 0.0008), 485 nm (Pearson r = 0.80, 95% CI 0.43–0.94, p = 0.0012) and 520 nm (Pearson r = 0.79, 95% CI 0.41–0.93, p = 0.0015). Supplementary Figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003. Molecular analysis of the hyperpigmented GBS isolates Molecular alterations within the CovS/R sites are presented in Fig. 3A. The alterations included a three-base-pair deletion (Val31) (isolate 001) and two nucleotide substitutions leading to Ala90Asp (isolate 013) and Arg119His (isolate 016), respectively. Further, in isolate 014 (both variants A and B), there was a nucleotide insertion leading to a truncated CovR at amino acid 206. In variant B of isolate 010, there was a 13-nucleotide deletion causing an abortive CovR (stop at amino acid 11) and an IS1381 inserted between the ribosome-binding site and the CovR binding site upstream of the covR gene. In isolate 012, the analysis of the promotor region of the cyl operon revealed a 19-base deletion from −295 to −277, from the cylX start codon, overlapping the CovR binding site (−281 to −252) (Fig. 3B). No covS/R variations were found in hyperpigmented isolates 002, 003, 008 and 015B. Transcriptional analysis and phenotypic tests of virulence factors
201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226
We identified 10 GBS hyperpigmented isolates obtained from 10 individuals. In seven patients, there was only one phenotype. In three patients, the hyperpigmented isolate was accompanied by a phenotypically different variant (i.e. variant A and B). In two of them (010, 015), one variant was considered non-hyperpigmented (variant A), whereas in one isolate (014) both variants showed an orange-reddish pigment. However, in one variant of 014 (variant B), the pigment was more reddish and the absorbance more intense (Fig. 1). PFGE analysis suggested that variants of the three isolates (010, 014, 015) were indistinguishable (supplementary file, Fig. S1). Hence, 13 isolates in total were included in this study. The source and characterization of the isolates is presented in Table 1. Three isolates were obtained from patients who had no disease manifestation (i.e. colonization) and seven isolates from sites in patients with a clinical syndrome. Disease manifestation varied from nonsevere (conjunctivitis, tonsillopharyngitis) to very severe (necrotizing fasciitis). None of the capsular serotypes was significantly associated with hyperpigmentation. The results of MSLT analysis are presented in Table 1. Three isolates were single locus variants of other sequence types (i.e. isolate 008 ST335 = variant of ST19, isolates 010A/B ST255 = variants of ST6, isolate 016 ST9 = variant of ST10). Apart from isolate 015A/B (singleton ST130), 10 isolates belonged to clonal complex (CC) 19, and one to CC17. All isolates were susceptible to penicillin, erythromycin and clindamycin. Supplementary Figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003.
We then quantified the transcripts of selected virulence genes in isolates harboring covS/R modifications and their variants (isolates 001, 010A and B, 013, 014A and B, 016) as well as that of NEM316 (control). Transcription of the cpsG gene was low in all isolates. The cylE gene was overexpressed in all isolates, except in variant A of isolate 010, which had a functional CovR. It was also significantly overexpressed when compared with cfb and cpsG in all isolates but 010A and 014A (Fig. 4). The expression patterns of 010A and its hyperpigmented variant 010B, which has a truncated CovR, were in agreement with the observed phenotypes of the isolates (Fig. 4 and supplementary file, Fig. S3), confirming the role of the CovS/R system as observed previously (Sendi et al., 2009). Variant B of isolate 014, which harbored the same CovR mutation as that of 014A, showed an overexpression of the cylE gene compared with the cfb and cpsG genes. Surprisingly, the cfb gene was overexpressed in variant 014B (Fig. 4). These analyzed transcript levels were in agreement with phenotypic profiles (supplementary file, Fig. S3). Supplementary Figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2014.05.003. Discussion In this study, we report the molecular and phenotypic characterization of a collection of clinical GBS isolates showing pronounced
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
227
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246
247
248 249 250 251 252 253 254 255 256 257 258 259 260 261
262
263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282
283
284 285
G Model IJMM 50826 1–8 4
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
Fig. 1. Photos of the colonies, the DMSO-washed pellets and the absorbance spectra of the GBS isolates.
286 287 288 289 290 291 292 293 294 295 296 297 298 299
pigmentation. To the best of our knowledge, the term hyperpigmentation is not precisely defined. The selection criteria applied in this study were based on phenotypic characteristics and analyses of pigment absorbance spectra. Each so-called hyperpigmented isolate included in this study had a pigment absorbance that was at least more than one log higher than those obtained from NEM316 (Fig. 1). Although more in-depth (e.g. chemical and physical) analysis is needed to define ‘hyperpigmented’ isolates, we believe that our approach appears reasonable and practicable for other research laboratories. The hyperpigmented isolates were collected from different anatomical sites and various patients. They were found as colonizers of the urogenital tract; in nonsevere diseases, such as tonsillopharyngitis and conjunctivitis; and in life-threatening
infections, such as necrotizing fasciitis. Thus, hyperpigmentation in clinical isolates is not uniformly associated with severe invasive disease. Recent advances in the understanding of the link between pigment production and -h/c activity in GBS have been achieved. Whidbey et al. (Whidbey et al., 2013) showed that the pigment consists of an ornithine rhamnolipid compound, and that this pigment has hemolytic and cytolytic activity. Consistent with these and previous results (Nizet et al., 1996), we found an overall good correlation between pigmentation and hemolytic activity (Fig. 2B). This was in particular obvious with isolates that revealed high absorbance values (i.e. 001, 008, 012, 010B and 014B). However, because we did not quantify the amount of the ornithine rhamnolipid compound, a direct correlation between pigment and
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
300 301 302 303 304 305 306 307 308 309 310 311 312 313
G Model
ARTICLE IN PRESS
IJMM 50826 1–8
A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
5
Table 1 Characterization of selected clinical GBS isolates. Isolate
Specimen
Patient
Clinical manifestation
Age
Sex
Biopsy Cervix swab Hip joint fluid Cervix swab Eye swab
74 22 88 20 1 month
M F M F M
Prosthetic joint infection No disease, colonization Septic arthritis No disease, colonization Conjunctivitis
Urine Biopsy Vaginal swab
64 77 55
F M F
Urine
65
M
Urinary tract infectionb Necrotizing fasciitis Bleeding from uterus myomatosus No disease, colonizationc
Throat swab
4
M
Tonsillopharnyngitis
Serotype
ST
MIC (E-test) P S ≤ 0.25
001 002 003 008 010Aa 010Ba 012 013 014Aa 014Ba 015Aa 015Ba 016
V III Ib III Ib Ib III III II II IX IX Ib
12 19 8 335 255 255 28 17 19 19 130 130 9
E R > 0.25
0.094 0.064 0.125 0.047 0.064 0.064 0.047 0.064 0.023 0.023 0.023 0.023 0.064
S ≤ 0.25
CLI R > 0.5
0.064 0.064 0.032 0.064 0.047 0.047 0.047 0.032 0.016 0.016 0.032 0.032 0.032
S ≤ 0.5
R > 0.5 0.094 0.094 0.094 0.094 0.25 0.25 0.064 0.064 0.047 0.047 0.125 0.125 0.094
M, male; F, female; ST, sequence type determined by MLST; MIC, minimal inhibitory concentration determined by E test; P, penicillin; E, erythromycin; CLI, clindamycin. a A and B correspond to clonally identical but phenotypically different variants: A, variant; B, hyperpigmented. b Polymicrobial infection. c Urine after bladder reconstruction and radiation after cancer.
314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333
hemolytic activity cannot be made. In addition, we found reddish colonies (e.g. 003, 015B) that did not reveal high absorbance values of the pigment extract or hemolytic activity. These discrepancies may be explained by variations in the chromophore part of the molecule, which would not affect its hemolytic property. Thus, pigment and hemolytic activity are often coupled, but this phenomenon cannot be applied to all clinical isolates. Moreover, results suggesting uniform correlation between pigment and hemolytic activity should be interpreted with caution because -h/c is an unstable cell-wall-associated toxin, and pure extraction of both pigment and -h/c is technically challenging. However, our study is the first to characterize pigmentation and hemolytic activity in a clinical collection of isolates with different genetic backgrounds and physiological features. In three specimens, hyperpigmented isolates (010B, 014B and 015B) were found together with an accompanying variant. Interestingly, in isolate 014, both variants were considered ‘hyperpigmented’, though they showed different levels of pigmentation. This phenomenon of clonally identical but phenotypically different GBS variants has been described previously in isolates obtained
from patients with severe disease (Sigge et al., 2008; Sendi et al., 2009). The hyperpigmented variants showed decreased CAMP and capsule production and carried mutations in the regulatory gene covR. However, in this study, the previously described characteristics were found in only one (010A and B) of the three paired variants. While the capsule was less expressed in all three hyperpigmented variants, the CAMP reaction was not decreased in isolates 014B and 015B (supplementary file, Fig. S3). Thus, although phenotypic characterization was performed in only three paired variants, it appears that in hyperpigmented isolates, capsule production is decreased. In contrast, an association between hyperpigmentation and a decrease in the CAMP reaction was observed in only one of the three isolates. We investigated a possible role of the covS/R system in hyperpigmented isolates and found that in five isolates, the covR sequence was mutated. These mutations caused truncation of CovR in two isolates (variant B of isolate 010 and both variants [A and B] of isolate 014) and a single amino acid deletion and substitution in the remaining three isolates (Val31, Ala90Asp and Arg119His in isolates 001, 013 and 016, respectively) (Fig. 3A). In addition, a partial
Fig. 2. (a) Hemolytic activity of GBS isolates. The values obtained at a dilution of 10−4 were compared with those obtained from 1% sheep blood treated with a 0.1% sodium dodecyl sulphate solution. (b) Association between absorbance intensity of the pigment at 455, 485 and 520 nm and hemolytic activity.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353
G Model IJMM 50826 1–8 6
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
Fig. 3. (a) Alignment of the CovR amino acid sequences from the isolates harboring mutations and highlights of the different residues compared with sequence NEM316 (accession number AL766852.1) (ClustalW). (b) Alignment of the cylX promoter region of isolate 012 and NEM316 (accession number AL766846.1). The CovR binding region (−281, −252 from ATG starting codon) proposed by Lamy et al. (2004) (Clustal W) is in bold and underlined.
354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
deletion in the CovR binding site (Pcyl region) in isolate 012 was detected (Fig. 3B). All of these mutations, except Val31 in isolate 001 (Sendi et al., 2009), are here reported for the first time in GBS. The functional consequences of the amino acid substitutions found in our study are unknown. They may be in line with the in vitro findings described by Lin et al. (2009) in GBS and Horstmann et al. (2011) in Group A Streptococcus (GAS). Promoter binding of CovR required phosphorylation of the aspartate residue at position 53. Phosphorylation of threonine at position 65 prevented CovR from binding to DNA (Lin et al., 2009). Expression of -h/c may thereby be de-repressed. Considering the homology and the similar functions between GAS and GBS CovR, a parallelism between the two structures can be speculated. The substitutions observed in the hyperpigmented isolates most likely affect the receiver domain of the CovR protein, probably interfering with the efficiency of phosphorylation by CovS. In particular, Arg119 substitution has been repeatedly described in GAS isolates (Horstmann et al., 2011). To confirm a role for the detected mutations in the regulatory pattern of our hyperpigmented isolates, we performed a
transcription analysis of the cfb (CAMP), cpsG (capsule) and cylE genes (hyperpigmentation). In hyperpigmented isolates (001, variant B of 010, 013, variants A and B of 014, and 016), CovR mutations were associated with the overexpression of the cylE gene and the downregulation of the cpsG gene. The findings in isolate 014 are consistent with the phenotypic observation that both variants showed an orange-reddish pigment. The downregulation of the cfb gene was not observed in variant B of isolate 014 or in isolate 016 (supplementary file, Fig. S3). The cfb gene was upregulated in variant A of isolate 010. Thus, in clinical hyperpigmented isolates with a mutated CovR, transcript analysis was uniformly consistent with cylE overexpression and cpsG downregulation, but downregulation of cfb (CAMP) was not found in all isolates. This parallels the abovedescribed phenotypic observations in the three isolates with paired variants (010, 014, 015). Considering that -h/c and the capsule are major virulence components, this investigation on clinical isolates supports the role of the CovS/R system in the regulation of these factors involved in GBS pathogenesis. Of note, CAMP appears not to be essential for systemic virulence in GBS (Hensler et al., 2008).
Fig. 4. cfb, cpsG and cylE gene expression was analyzed in the isolates harboring mutations in the covS/R sequence. For each isolate, we performed an absolute quantification of the mRNA transcripts normalized to the expression level of the gyrA housekeeping gene. The level of expression of the cylE gene was compared with that of the cfb and the cpsG genes by t-test.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
G Model IJMM 50826 1–8
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444
445
446 447 448 449 450 451 452 453
Our study has limitations. Hyperpigmented isolates were looked for on the basis of phenotypic characteristics, but in contrast to accepted characteristics of phenotypic variants in other species (e.g. small colony variants of Staphylococcus aureus (Sendi and Proctor, 2009)), there are no uniformly accepted characteristics to discriminate these GBS isolates. We focused on reddish isolates, and (photo) documented applied criteria that appeared reasonable to us. We also used several criteria because one criterion would be unreliable and no correlation can be made on visual parameters (e.g. phenotype on an agar plate versus product after DMSO treatment). However, these criteria may be unpractical for screening in a routine diagnostic laboratory. Selecting colonies for further investigation on the basis of phenotype through identification by laboratory staff may be an insensitive method. Also special media may be required (e.g. Columbia blood agar containing proteose peptone 3 and starch). Thus, it is possible that an unknown number of hyperpigmented isolates were missed during the observation period. Consequently, no firm incidence rate on hyperpigmented isolates can be given. The selection of 10 isolates during a 4.5-year period was found in an institute that registers approximately 350–400 GBS isolates per year. Yet, we have presented the largest published series of clinical GBS showing hyperpigmentation. Another limitation includes the fact that the array of genes co-regulated by CovR is wider than the number of genes analyzed in this study. On the other hand, the core CovS/R regulon comprises the genes selected in this study (Jiang et al., 2005). Finally, hyperpigmentation may be associated with mutations outside of the CovS/R pathways, since we found four isolates without mutations in the covS/R genes and their binding sites. Analyzing all possible genes potentially responsible for hyperpigmented phenotypes is, however, beyond the scope of this study. In conclusion, clinical hyperpigmented isolates are associated with increased hemolytic activity in vitro, although hyperpigmentation in GBS isolates is not strictly related to invasive diseases and mutations within the covS/R genes. In three of four isolates without genetic alterations, no associated clinical syndrome was present. In 6 of 10 isolates, mutations were found, but only two of them were obtained from patients with acute severe infections (periprosthetic joint infection and necrotizing fasciitis). The alterations found within the CovS/R system of hyperpigmented isolates were consistently correlated to increased cylE and decreased cpsG (capsule) expression. These findings can suggest a correlation between the alteration of the CovS/R system and the development of invasive disease, as previously postulated for isogenic mutants (Lamy et al., 2004; Liu et al., 2004; Jiang et al., 2005; Whidbey et al., 2013). However, covR mutations were also found in patients without clinical syndromes. Adding to the puzzle are the unpredicted results in the regulatory pattern of the analyzed genes (e.g. cfb [CAMP]) compared with in vitro investigations of isogenic mutants. This highlights the necessity of performing additional clinical studies to further elucidate the complex GBS regulatory network and its pathogenesis.
Acknowledgments This study was presented in part at the 7th International Conference on Gram-Positive Microorganisms, Montecatini Terme, Tuscany, Italy (P165). This work is supported by the Velux Stiftung, Zurich, Switzerland. We thank Drs. Andrea Endimiani and Sara Droz, and Gabriela Reichen, for critical discussion and technical support. The manuscript was edited by Barbara Every, BioMedical Editor, St. Albert, Alberta, Canada.
7
References Buchanan, J.T., Stannard, J.A., Lauth, X., Ostland, V.E., Powell, H.C., Westerman, M.E., Nizet, V., 2005. Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development. Infection and Immunity 73, 6935–6944. CLSI, 2010. Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing Twentieth Informational Supplement M100-S20. Clinical and Laboratory Standards Institute, Wayne, PA. Doran, K.S., Chang, J.C., Benoit, V.M., Eckmann, L., Nizet, V., 2002. Group B streptococcal beta-hemolysin/cytolysin promotes invasion of human lung epithelial cells and the release of interleukin-8. Journal of Infectious Diseases 185, 196–203. Florindo, C., Ferreira, R., Borges, V., Spellerberg, B., Gomes, J.P., Borrego, M.J., 2012. Selection of reference genes for real-time expression studies in Streptococcus agalactiae. Journal of Microbiological Methods 90, 220–227. Gibson, R.L., Nizet, V., Rubens, C.E., 1999. Group B streptococcal beta-hemolysin promotes injury of lung microvascular endothelial cells. Pediatric Research 45, 626–634. Gottschalk, B., Broker, G., Kuhn, M., Aymanns, S., Gleich-Theurer, U., Spellerberg, B., 2006. Transport of multidrug resistance substrates by the Streptococcus agalactiae hemolysin transporter. Journal of Bacteriology 188, 5984–5992. Hensler, M.E., Quach, D., Hsieh, C.J., Doran, K.S., Nizet, V., 2008. CAMP factor is not essential for systemic virulence of Group B Streptococcus. Microbial Pathogenesis 44, 84–88. Horstmann, N., Sahasrabhojane, P., Suber, B., Kumaraswami, M., Olsen, R.J., Flores, A., Musser, J.M., Brennan, R.G., Shelburne 3rd, S.A., 2011. Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis. PLoS Pathogens 7, e1002311. Imperi, M., Pataracchia, M., Alfarone, G., Baldassarri, L., Orefici, G., Creti, R., 2010. A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae. Journal of Microbiological Methods 80, 212–214. Jiang, S.M., Cieslewicz, M.J., Kasper, D.L., Wessels, M.R., 2005. Regulation of virulence by a two-component system in group B streptococcus. Journal of Bacteriology 187, 1105–1113. Jones, N., Bohnsack, J.F., Takahashi, S., Oliver, K.A., Chan, M.S., Kunst, F., Glaser, P., Rusniok, C., Crook, D.W., Harding, R.M., Bisharat, N., Spratt, B.G., 2003. Multilocus sequence typing system for group B Streptococcus. Journal of Clinical Microbiology 41, 2530–2536. Lamy, M.C., Zouine, M., Fert, J., Vergassola, M., Couve, E., Pellegrini, E., Glaser, P., Kunst, F., Msadek, T., Trieu-Cuot, P., Poyart, C., 2004. CovS/CovR of group B Streptococcus: a two-component global regulatory system involved in virulence. Molecular Microbiology 54, 1250–1268. Lin, W.J., Walthers, D., Connelly, J.E., Burnside, K., Jewell, K.A., Kenney, L.J., Rajagopal, L., 2009. Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR. Molecular Microbiology 71, 1477–1495. Liu, G.Y., Doran, K.S., Lawrence, T., Turkson, N., Puliti, M., Tissi, L., Nizet, V., 2004. Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense. Proceedings of the National Academy of Sciences of the United States of America 101, 14491–14496. Liu, G.Y., Nizet, V., 2004. Extracellular virulence factors of group B Streptococci. Frontiers in bioscience: A Journal and Virtual Library 9, 1794–1802. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Maisey, H.C., Doran, K.S., Nizet, V., 2008. Recent advances in understanding the molecular basis of group B Streptococcus virulence. Expert Reviews in Molecular Medicine 10, e27. Merritt, K., Jacobs, N.J., 1978. Characterization and incidence of pigment production by human clinical group B streptococci. Journal of Clinical Microbiology 8, 105–107. Murray, B.E., Singh, K.V., Heath, J.D., Sharma, B.R., Weinstock, G.M., 1990. Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology 28, 2059–2063. Nizet, V., Gibson, R.L., Chi, E.Y., Framson, P.E., Hulse, M., Rubens, C.E., 1996. Group B streptococcal beta-hemolysin expression is associated with injury of lung epithelial cells. Infection and Immunity 64, 3818–3826. Pritzlaff, C.A., Chang, J.C., Kuo, S.P., Tamura, G.S., Rubens, C.E., Nizet, V., 2001. Genetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Molecular Microbiology 39, 236–247. Ring, A., Braun, J.S., Pohl, J., Nizet, V., Stremmel, W., Shenep, J.L., 2002. Group B streptococcal beta-hemolysin induces mortality and liver injury in experimental sepsis. Journal of Infectious Diseases 185, 1745–1753. Rosa-Fraile, M., Rodriguez-Granger, J., Haidour-Benamin, A., Cuerva, J.M., Sampedro, A., 2006. Granadaene: proposed structure of the group B Streptococcus polyenic pigment. Applied and Environmental Microbiology 72, 6367–6370. Sendi, P., Johansson, L., Dahesh, S., Van-Sorge, N.M., Darenberg, J., Norgren, M., Sjolin, J., Nizet, V., Norrby-Teglund, A., 2009. Bacterial phenotype variants in group B streptococcal toxic shock syndrome. Emerging Infectious Diseases 15, 223–232. Sendi, P., Johansson, L., Norrby-Teglund, A., 2008. Invasive group B Streptococcal disease in non-pregnant adults: a review with emphasis on skin and soft-tissue infections. Infection 36, 100–111. Sendi, P., Proctor, R.A., 2009. Staphylococcus aureus as an intracellular pathogen: the role of small colony variants. Trends in Microbiology 17, 54–58.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
454
455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537
G Model IJMM 50826 1–8 8 538 539 540 541 542 543 544
ARTICLE IN PRESS A. Lupo et al. / International Journal of Medical Microbiology xxx (2014) xxx–xxx
Sigge, A., Schmid, M., Mauerer, S., Spellerberg, B., 2008. Heterogeneity of hemolysin expression during neonatal Streptococcus agalactiae sepsis. Journal of Clinical Microbiology 46, 807–809. Slotved, H.C., Elliott, J., Thompson, T., Konradsen, H.B., 2003. Latex assay for serotyping of group B Streptococcus isolates. Journal of Clinical Microbiology 41, 4445–4447. Spellerberg, B., Pohl, B., Haase, G., Martin, S., Weber-Heynemann, J., Lutticken, R., 1999. Identification of genetic determinants for the hemolytic activity of
Streptococcus agalactiae by ISS1 transposition. Journal of Bacteriology 181, 3212–3219. Tapsall, J.W., 1986. Pigment production by Lancefield-group-B streptococci (Streptococcus agalactiae). Journal of Medical Microbiology 21, 75–81. Whidbey, C., Harrell, M.I., Burnside, K., Ngo, L., Becraft, A.K., Iyer, L.M., Aravind, L., Hitti, J., Waldorf, K.M., Rajagopal, L., 2013. A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta. Journal of Experimental Medicine 210, 1265–1281.
Please cite this article in press as: Lupo, A., et al., Phenotypic and molecular characterization of hyperpigmented group B Streptococci. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.05.003
545 546 547 548 549 550 551 552
