LETTERS

Rennes. This strain, characterized (C. Plainvert, A. Bouvet); Université de infections in injecting drug users in the by PFGE pattern 44-A1, represented Rennes1, Rennes (P.-Y. Donnio, S. Kayal); UK, 2003–2004. Clin Microbiol Infect. 2008;14:1002–9. DOI: 10.1111/j.1469- Cellule de l’Institut National de Veille Sani- 22/25 tetracycline resistant GAS iso- 0691.2008.02076.x lates and 30% of the 72 GAS isolates taire en Région Ouest, Rennes (P. Loury, 10. Sierra JM, Sanchez F, Castro P, Salvado identifi ed at the hospital in Pontchail- A. Briand); and de l’Agence Régionale de M, de la Red G, Libois A, et al. Group A lou in 2009. Locally, emergence of Santé de Bretagne, Rennes (D. Huguenet) streptococcal infections in injection drug users in Barcelona, Spain: epidemiologic, the 44-A1 clone led to the dramatic DOI: 10.3201/eid1702.101022 clinical, and microbiologic analysis of 3 increase of GAS tetracycline resis- clusters of cases from 2000 to 2003. Med- tance, from 17% in 2008 to 35% in icine (Baltimore). 2006;85:139–46. DOI: References 2009. emm44 GAS strains, which 10.1097/01.md.0000224707.24392.52 share identical 5′emm sequences with 1. Factor SH, Levine OS, Schwartz B, Har- Address for correspondence: Anne Cady, previously designated M/ emm61 rison LH, Farley MM, McGeer A, et al. Service de Bactériologie–Virologie et Hygiène strains (5), have mainly been isolated Invasive group A streptococcal disease: risk factors for adults. Emerg Infect Dis. Hospitalière, CHU Pontchaillou, 35033 in Asia from throat and skin speci- 2003;9:970–7. Rennes CEDEX, France; email: anne.cady@ mens (6,7). They were rarely reported 2. Lamagni TL, Darenberg J, Luca-Harari chu-rennes.fr as responsible for invasive infections B, Siljander T, Efstratiou A, Henriques- in France or other parts of the world Normark B, et al. Epidemiology of severe Streptococcus pyogenes disease in Europe. (5,8). Polyclonal and emm25 and J Clin Microbiol. 2008;46:2359–67. DOI: emm83 monoclonal GAS outbreaks 10.1128/JCM.00422-08 have been recently described among 3. Mihaila-Amrouche L, Bouvet A, Lou- drug users in Switzerland, the United binoux J. Clonal spread of emm type 28 isolates of Streptococcus pyogenes that Kingdom, and Spain (9,10) without are multiresistant to antibiotics. J Clin Mi- robust evidence of enhanced viru- crobiol. 2004;42:3844–6. DOI: 10.1128/ Surface Layer lence of the causative GAS strains. JCM.42.8.3844-3846.2004 In the outbreak we report, skin infec- 4. Tenover FC, Arbeit RD, Goering RV, Protein A Variant of Mickelsen PA, Murray BE, Persing DH, tions might be a leading cause of bac- et al. Interpreting chromosomal DNA Clostridium diffi cile terial transmission between people restriction patterns produced by pulsed- PCR-Ribotype 027 living in poor hygienic conditions fi eld gel electrophoresis: criteria for and overcrowded spaces. bacterial strain typing. J Clin Microbiol. To the Editor: Rates and severity 1995;33:2233–9. 5. Johnson DR, Kaplan EL, VanGheem A, of Clostridium diffi cile infection (CDI) Acknowledgments Facklam RR, Beall B. Characterization have recently increased worldwide We thank the local health authorities, of group A streptococci (Streptococcus and correlate with dissemination of pyogenes): correlation of M-protein and hypervirulent epidemic strains desig- the Institute for Public Health Surveil- emm-gene type with T-protein agglutina- lance, and the nurses working in social tion pattern and serum opacity factor. J nated PCR-ribotype 027. CDI caused centers for their helpful collaboration. We Med Microbiol. 2006;55:157–64. DOI: by this PCR-ribotype is characterized also thank Gislène Collobert and Gérald 10.1099/jmm.0.46224-0 by strong toxin A and B production, 6. Koh EH, Kim S, Lee NY. Decrease of presence of binary toxin genes, and, Touak for excellent technical assistance erythromycin resistance in group A strep- and Lucie Donnio for correcting the Eng- tococci by change of emm distribution. usually, a high level of resistance to lish in this manuscript. Jpn J Infect Dis. 2008;61:261–3. fl uoroquinolones (1). 7. Sagar V, Kumar R, Ganguly NK, The mechanisms by which C. dif- Chakraborti A. Comparative analysis of fi cile colonizes the gut during infection Anne Cady,1 Céline Plainvert,1 emm type pattern of group A streptococ- cus throat and skin isolates from India and are poorly understood. In addition to Pierre-Yves Donnio, the toxins, surface protein components Pascaline Loury, their association with closely related SIC, a streptococcal virulence factor. BMC Mi- are undoubtedly involved. In particu- Didier Huguenet, Alain Briand, crobiol. 2008;8:150. DOI: 10.1186/1471- lar, the surface layer (S-layer) medi- Matthieu Revest, Samer Kayal, 2180-8-150 ates adhesion to enteric cells (2), but and Anne Bouvet 8. Luca-Harari B, Darenberg J, Neal S, Sil- jander T, Strakova L, Tanna A, et al. Clini- other functions have been proposed Author affi liations: Centre Hospitalier Uni- cal and microbiological characteristics of for this S-layer structure: it may act versitaire Pontchaillou, Rennes, France (A. severe Streptococcus pyogenes disease in as a molecular sieve, protect against Cady, P.-Y. Donnio, M. Revest, S. Kayal); Europe. J Clin Microbiol. 2009;47:1155– parasitic attack, or be a mechanism University Paris Descartes, Paris, France 65. DOI: 10.1128/JCM.02155-08 9. Lamagni TL, Neal S, Keshishian C, Hope to evade the host immune system (3). V, George R, Duckworth G, et al. Epi- 1These authors contributed equally to this Furthermore, the C. diffi cile S-layer is demic of severe Streptococcus pyogenes article. the predominant surface antigen and is

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 2, February 2011 317 LETTERS among the main potential candidates whereas CD196, LUMC46, and A422 and the output was used for construc- for multicomponent vaccines against were susceptible to both drugs. tion of the phylogenetic tree by Tree- CDI (4,5). Composed of 2 major com- The slpA genes of all strains View version 1.6.6 (http://en.bio-soft. ponents, the C. diffi cile S-layer has were amplifi ed by PCR mapping. net/tree/TreeView.html). All PCR- high and low molecular weight pro- Nine primers were designed on the ribotype 027 strains showed the same teins (HMW and LMW, respectively), slpA region to obtain 10 overlapping slpA gene nucleotide sequence. The which are formed from the posttrans- PCR products. The positions of the slpA precursor encoded by this gene lational cleavage of a single precursor, primers on the reference sequence contained a signal peptide, and its surface layer protein A (slpA) (6). Dif- FN545816 were 3161991–31612012, cleavage site was located between ferent variants of the slpA gene have 3162346–3162365, 3162728–3162746, aa 24 and aa 25. The cleavage of the been identifi ed in C. diffi cile (7). 3162746–3162728, 3163514–3163495, slpA precursor into LMW and HMW The complete genome sequenc- 3164222–3164205, 3163264–3163284, proteins was predicted between aa es of 2 C. diffi cile PCR-ribotype 3163284–3163264, and 3164518– 342 and aa 343 (N terminal to an Ala 027 strains (CD196, a nonepidemic 3164499. Target amplifi cation was amino acid residue and C-terminal to strain isolated in France in 1985, and performed by an initial denaturation a consensus motif Thr-Lys-Ser). The R20291, isolated from an outbreak at 94°C for 5 min, then 30 cycles of molecular masses of the LMW and in Stoke Mandeville, UK, in 2006) 94°C for 1 min, 50°C for 1 min, and HMW proteins were 33.871 kDa and have been recently deposited in Gen- 72°C for 1 min. Sequence assembly 44.174 kDa, respectively. These pro- Bank (accession nos. FN538970 and was performed by using DNAStar La- tein sizes were confi rmed by sodium FN545816, respectively) (8). We ana- sergene version 8.0 software (DNA- dodecyl sulfate polyacrylamide gel lyzed the slpA gene of these strains Star, Madison, WI, USA). The protein electrophoresis, after a low pH gly- by using the National Center for analysis was performed by using the cine extraction (data not shown). The Biotechnology Information BLAST SignalP 3.0 server (www.cbs.dtu.dk/ phylogenetic tree (Figure), obtained server (www.ncbi.nlm.nih.gov/blast) services/SignalP/) and the ExPASy by comparison with the amino acid and the European Bioinformatics In- Proteomics server (www.expasy.ch/ sequences of other PCR ribotypes stitute ClustalW server (www.ebi. tools/pi_tool.html). Amino acid com- (6), showed that C. diffi cile strain 027 ac.uk/clustalw). Both strains showed parisons were accomplished by using slpA was strongly related (identity a new and identical slpA nucleotide ClustalW (www.ebi.ac.uk/clustalw), 89%) to that of strains belonging to sequence. To determine if the new variant was conserved among PCR-ri- botype 027 strains, we characterized 8 additional epidemic strains belonging to this PCR-ribotype that were isolat- ed in different geographic regions and years and showed different patterns of resistance to erythromycin and moxi- fl oxacin. Three strains, AI13, AII6, and AIII8, were isolated in 3 hospitals in Belgium during a European pro- spective study conducted in 2005 (9). C. diffi cile DI12 was isolated in Ire- land during the same study. C. diffi cile GII7 and LUMC46 were isolated in the Netherlands in 2005 and 2008, re- spectively. C. diffi cile M43 and A422 were isolated in Calgary (Canada) in 2001 from 2 outbreaks. Figure. Phylogenetic tree based on the alignment of the surface layer protein A amino Six strains were resistant to eryth- acid sequence of Clostridium diffi cile 027 (GenBank accession no. CBE06198) with those romycin (MICs >256 mg/L) and mox- of PCR-ribotypes 001, 002, 005, 010, 012, 014, 017, 031, 046, 054, 066, 078, 092, and ifl oxacin (MICs 12–256 mg/L). AIII8 094 (GenBank accession nos. AAZ05957, AAZ05964, AAZ05968, AAZ05974, AAZ05975, was resistant to erythromycin (MIC AAZ05984, AAZ05988, AAZ05989, AAZ05980, AAZ05972, AAZ05986, AAZ05994, AAZ05982, and AAZ05991, respectively). The phylogram was generated by using >256 mg/L) and intermediately resis- TreeView version 1.6.6 (http://en.bio-soft.net/tree/TreeView.html). The branch lengths are tant moxifl oxacin (MIC = 6 mg/L), scaled in proportion to the extent of the change per position, as indicated by the scale bar.

318 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 2, February 2011 LETTERS the epidemic PCR-ribotype 001. In 2. Calabi E, Calabi F, Phillips AD, Fairweath- Introduction particular, the identity between the er NF. Binding of Clostridium diffi cile surface layer proteins to gastrointestinal of Japanese 2 PCR ribotypes was 100% for the tissues. Infect Immun. 2002;70:5770–8. HMW proteins and 77% for the LMW DOI: 10.1128/IAI.70.10.5770-5778.2002 Encephalitis Virus proteins. 3. Sára M, Sleytr UB. S-layer proteins. J Bac- Genotype I, India This study provides convincing teriol. 2000;182:859–68. DOI: 10.1128/ JB.182.4.859-868.2000 evidence that the S-layer is well con- 4. Pantosti A, Cerquetti M, Viti F, Ortisi G, To the Editor: Seasonal out- served in C. diffi cile PCR-ribotype Mastrantonio P. Immunoblot analysis of breaks of fatal acute encephalitis syn- 027 strains and has high identity with serum immunoglobulin G response to sur- drome (AES) occur regularly in sever- the slpA of the epidemic PCR-ribo- face proteins of Clostridium diffi cile in pa- al parts of India. Japanese encephalitis tients with antibiotic-associated diarrhea. type 001. Because C. diffi cile PCR- J Clin Microbiol. 1989;27:2594–7. virus (JEV) has been the major and ribotypes 027 and 001 are the most 5. Ausiello CM, Cerquetti M, Fedele G, Spen- consistent cause of these outbreaks in frequently isolated strains from severe sieri F, Palazzo R, Nasso M, et al. Surface the Gorakhpur region of Uttar Pradesh CDIs across both North America and layer proteins from Clostridium diffi cile State, accounting for ≈10%–15% of induce infl ammatory and regulatory cy- Europe (9,10), the result obtained sug- tokines in human monocytes and dendritic total AES cases annually (1–3). In gests that the S-layer of these virulent cells. Microbes Infect. 2006;8:2640–6. India, vaccinations against Japanese strains presents peculiar and common DOI: 10.1016/j.micinf.2006.07.009 encephalitis (JE) are administered in characteristics that could be an advan- 6. Eidhin DN, Ryan AW, Doyle RM, Walsh areas where the disease is hyperen- JB, Kelleher D. Sequence and phylo- tage for these bacteria during the in- genetic analysis of the gene for surface demic, including Gorakhpur, and AES fection process. layer protein, slpA, from 14 PCR ribo- cases are regularly investigated to types of Clostridium diffi cile. J Med Mi- clarify the effects of vaccination. Cur- crobiol. 2006;55:69–83. DOI: 10.1099/ Acknowledgments rently, >2,000 patients with AES are jmm.0.46204-0 We thank the following participants 7. Fagan RP, Albesa-Jové D, Qazi O, Svergun admitted each year to Baba Raghav of the 2005 European Prospective Study DI, Brown KA, Fairweather NF. Structur- Das Medical College, Gorakhpur. on Clostridium diffi cile: M. Delmé, D. al insights into the molecular organization JEV is classifi ed into 5 genotypes. of the S-layer from Clostridium diffi cile. Drudy, and E. Kuijper for providing strains Genotype III (GIII) is widely distrib- Mol Microbiol. 2009;71:1308–22. DOI: AI13, AII6, AIII8, DI12, and GII7; E. Kui- 10.1111/j.1365-2958.2009.06603.x uted in Asian countries, including jper for providing strain LUMC46; and T. 8. Stabler RA, He M, Dawson L, Martin Japan, South Korea, the People’s Re- Louie for providing strains M43 and A422. M, Valiente E, Corton C, et al. Compara- public of China, Taiwan, Vietnam, the tive genome and phenotypic analysis of We also thank Tonino Sofi a for editing the Philippines, India, Nepal, and Sri Lan- Clostridium diffi cile 027 strains provides manuscript. insight into the evolution of a hypervirulent ka (4). However, during the past de- bacterium. Genome Biol. 2009;10:R102. cade, JEV GI has been introduced into This work was partially supported DOI: 10.1186/gb-2009-10-9-r102 South Korea, Thailand, and China and by the European Community project “The 9. Barbut F, Mastrantonio P, Delmée M, Bra- has replaced the GIII strains that had zier J, Kuijper E, Poxton I. European Study Physiological Basis of Hypervirulence in been circulating in Japan and Vietnam Clostridium diffi cile: A Prerequisite for Group on Clostridium diffi cile (ESGCD). Prospective study of Clostridium diffi cile during the mid-1990s (5). Until 2007, Effective Infection Control”—HEALTH- infections in Europe with phenotypic and all known JEV strains isolated in India F3-2008-223585. genotypic characterisation of the isolates. belonged to GIII (2–4,6). Clin Microbiol Infect. 2007;13:1048–57. DOI: 10.1111/j.1469-0691.2007.01824.x The JE-endemic Gorakhpur region Patrizia Spigaglia, 10. Cheknis AK, Sambol SP, Davidson DM, recorded a sudden increase in AES cas- Fabrizio Barbanti, Nagaro KJ, Mancini MC, Hidalgo-Arroyo es during September–November 2009. and Paola Mastrantonio GA, et al. Distribution of Clostridium dif- Clinical specimens collected from 694 fi cile strains from a North American, Eu- Author affi liations: Istituto Superiore di San- ropean, and Australian trial of treatment hospitalized patients were examined for ità, Rome, Italy for C. diffi cile infections: 2005–2007. An- JEV infection by JEV-specifi c immuno- aerobe. 2009;15:230–3. DOI: 10.1016/j. globulin M capture ELISA (7). Clinical DOI: 10.3201/eid1702.100355 anaerobe.2009.09.001 specimens comprising 115 (16.6%) ce- rebrospinal fl uid (CSF) specimens and References Address for correspondence: Paola Mastran- 114 (16.4%) serum specimens showed tonio, Department of Infectious, Parasitic, and 1. O’Connor JR, Johnson S, Gerding DN. recent JE infection among 158 (22.7%) Immune-mediated Diseases, Istituto Superiore Clostridium diffi cile infection caused by of the case-patients. di Sanità, 299 Viale Regina Elena 00161 the epidemic BI/NAP1/027 strain. Gas- All CSF specimens were pro- troenterology. 2009;136:1913–24. DOI: Rome, Italy; email: [email protected] 10.1053/j.gastro.2009.02.073 cessed for JEV genome detection by diagnostic reverse transcription–PCR

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