Carmen Tarradas

Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen Streptococcus suis: Identification of Virulent Clones and Potential Capsular Serotype Exchange

ABSTRACT Streptococcus suis is an important pathogen of pigs and occasionally causes serious human disease. However, little is known about the S. suis population structure, the clonal relationships between strains, the potential of particular clones to cause disease, and the relevance of serotype as a marker for epidemiology. Here we describe a multilocus sequence typing (MLST) scheme for S. suis developed in order to begin to address these issues. Seven housekeeping gene fragments from each of 294 S. suis isolates obtained from various S. suis diseases and from asymptomatic carriage representing 28 serotypes and nine distinct countries of origin were sequenced. Between 32 and 46 alleles per locus were identified, giving the ability to distinguish >1.6 × 1011 sequence types (STs). However only 92 STs were identified in this study. Of the 92 STs 18 contained multiple isolates, the most common of which, ST1, was identified on 141 occasions from six countries. Assignment of the STs to lineages resulted in 37 being identified as unique and unrelated STs while the remaining 55 were assigned to 10 complexes. ST complexes ST1, ST27, and ST87 dominate the population; while the ST1 complex was strongly associated with isolates from septicemia, meningitis, and arthritis, the ST87 and ST27 complexes were found to contain significantly higher numbers of lung isolates. In agreement with the observed distribution of disease-causing isolates of S. suis, most isolates previously characterized as of high virulence in porcine infection models belong to ST1, while isolates belonging to other STs appear to be less virulent in general. Finally nine STs were found to contain isolates of multiple serotypes, and many isolates belonging to the same serotypes were found to have very disparate genetic backgrounds. As well as highlighting that the serotype can often be a poor indicator of genetic relatedness between S. suis isolates, these findings suggest that capsular genes may be moving horizontally through the S. suis population.

Analysis of Genetic Diversity of Streptococcus suis Clinical Isolates from Pigs in Spain by Pulsed-Field Gel Electrophoresis

ABSTRACT Pulsed-field gel electrophoresis (PFGE) was used to investigate the diversity of Streptococcus suis isolates of various serotypes recovered from swine clinical samples in Spain. Capsular types 9 (64.9%) and 2 (14.8%) were the most frequently isolated serotypes followed by serotype 7 (5.9%) and serotype 8 (4.3%). The PFGE results of this study with 60 different pulsotypes indicate a great genetic diversity among the S. suis isolates, which is consistent with the broad distribution of S. suis in the swine population. Forty-five percent of the pulsotypes corresponded to single isolates, no pulsotype was common to all farms, and at least 3 different pulsotypes were isolated in 56% of herds in which more than 3 clinical isolates were analyzed. These results reveal a great diversity both between and within herds throughout the strains of S. suis studied, demonstrating that different strains of S. suis are associated with infection in pigs. Some pulsotypes were more frequently isolated and exhibited a wider distribution over herds than others, and were the unique or predominant strains in several herds, suggesting the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. Overall, the great genetic heterogeneity of the clinical strains of S. suis, the isolation of different strains within the same herd, and the predominance of particular strains in some herds are evidence that infection by S. suis is a dynamic process and reinforce the idea that the epidemiology of S. suis infection is very complex.

Distribution and Genetic Diversity of Suilysin in Streptococcus suis Isolated from Different Diseases of Pigs and Characterization of the Genetic Basis of Suilysin Absence

ABSTRACT Streptococcus suis is an economically important pathogen of pigs responsible for a variety of diseases including meningitis, septicemia, arthritis, and pneumonia, although little is known about the mechanisms of pathogenesis or virulence factors associated with this organism. Here, we report on the distribution and genetic diversity of the putative virulence factor suilysin, a member of the thiol-activated toxin family of gram-positive bacteria. On the basis of PCR analysis of over 300 isolates of S. suis, the suilysin-encoding gene, sly, was detected in 69.4% of isolates. However, sly was present in a considerably higher proportion of isolates obtained from cases of meningitis, septicemia, and arthritis (>80%) and isolates obtained from asymptomatic tonsillar carriage (>90%) than lung isolates associated with pneumonia (44%). With the exception of serotypes 1, 14, and 1/14, there was no strong correlation between the presence of suilysin and serotype. Analysis of the genetic diversity of suilysin by restriction fragment length polymorphism and sequence analysis found that the suilysin gene, where present, is highly conserved with a maximum of 1.79% diversity at the nucleotide level seen betweensly alleles. Assays of hemolytic activity and hybridization analysis provided no evidence for a second member of the thiol-activated toxin family in S. suis. Inverse PCR was used to characterize regions flanking sly, which in turn allowed the first characterization of the equivalent region in a strain lacking sly. Sequence comparison of these regions fromsly-positive (P1/7) and sly-negative (DH5) strains indicated that two alternative arrangements are both flanked by genes with highest similarity to haloacid dehalogenase-like hydrolases (5′ end) and putativeN-acetylmannosamine-6-phosphate epimerases (3′ end). However, sly appears to be completely absent from the alternative arrangement, and a gene of unknown function is located in the equivalent position. Finally, PCR analysis of multiplesly-positive and -negative strains indicated that these two alternative genetic arrangements are conserved among many S. suis isolates.

Overcoming function annotation errors in the Gram-positive pathogen Streptococcus suis by a proteomics-driven approach.

BackgroundAnnotation of protein-coding genes is a key step in sequencing projects. Protein functions are mainly assigned on the basis of the amino acid sequence alone by searching of homologous proteins. However, fully automated annotation processes often lead to wrong prediction of protein functions, and therefore time-intensive manual curation is often essential. Here we describe a fast and reliable way to correct function annotation in sequencing projects, focusing on surface proteomes. We use a proteomics approach, previously proven to be very powerful for identifying new vaccine candidates against Gram-positive pathogens. It consists of shaving the surface of intact cells with two proteases, the specific cleavage-site trypsin and the unspecific proteinase K, followed by LC/MS/MS analysis of the resulting peptides. The identified proteins are contrasted by computational analysis and their sequences are inspected to correct possible errors in function prediction.ResultsWhen applied to the zoonotic pathogen Streptococcus suis, of which two strains have been recently sequenced and annotated, we identified a set of surface proteins without cytoplasmic contamination: all the proteins identified had exporting or retention signals towards the outside and/or the cell surface, and viability of protease-treated cells was not affected. The combination of both experimental evidences and computational methods allowed us to determine that two of these proteins are putative extracellular new adhesins that had been previously attributed a wrong cytoplasmic function. One of them is a putative component of the pilus of this bacterium.ConclusionWe illustrate the complementary nature of laboratory-based and computational methods to examine in concert the localization of a set of proteins in the cell, and demonstrate the utility of this proteomics-based strategy to experimentally correct function annotation errors in sequencing projects. This approach also contributes to provide strong experimental evidences that can be used to annotate those proteins for which a Gene Ontology (GO) term has not been assigned so far. Function annotation correction would then improve the identification of surface-associated proteins in bacterial pathogens, thus accelerating the discovery of new vaccines in infectious disease research.

Risk factors associated with the antimicrobial resistance of staphylococci in canine pyoderma

This study reports the susceptibility to antimicrobial agents of staphylococci (n=105) isolated from dogs, and the factors associated with this resistance. The study animals were 23 healthy dogs (group A), 24 with first-time pyoderma (group B), and 27 with recurrent pyoderma that had undergone long-term antibiotic treatment (group C). Staphylococci were more commonly isolated from the pyoderma-affected than the healthy dogs (p<0.0001). Some 78% of the isolates were resistant to at least one antimicrobial agent. Resistance to amoxicillin-clavulanate, cephalosporins (OR 4.29, 95% CI [1.15, 16.3] respectively), enrofloxacin (OR 9.47, 95% CI [1.53, 58.5]) and ciprofloxacin (OR 79.7 95% CI [3.26, 1947.4]) was more common among group C isolates. Some 32% of all the isolates were multiresistant (MR) and 10.4% were methicillin-resistant (MRS). The probability of isolating MRS staphylococci in group C increased by a factor of four (95% CI [1.18, 17.9]) compared to A plus B. Multi-resistant (MR) isolates were obtained more commonly from urban than rural dogs (OR 3.79, 95% CI [1.09, 13.17]). All the MRS staphylococci encountered were obtained from urban dogs and more commonly from male dogs (p=0.07). This study shows that dogs bred in urban habitat, with a history of antibiotic therapy in the past year represents significant risk of being carriers of isolates resistant to methicillin (MRS) and other antimicrobials. These factors should be considered before applying an antimicrobial treatment in veterinary clinics.

A surface protein of Streptococcus suis serotype 2 identified by proteomics protects mice against infection

Streptococcus suis serotype 2 is a major Gram-positive swine pathogen, causing also zoonoses. We describe here the immunoprotective activity in an in vivo animal model of a serotype-2 cell wall protein, designated Sat, which was identified by a previously validated proteomics approach consisting of the protease digestion of live bacteria and the selective recovery of exposed domains, followed by LC/MS/MS analysis. Increased survival rate (80%) and decreased bacterial burden were observed in mice immunized with a recombinant Sat fragment, suggesting that this protein is a potential vaccine candidate against serotype-2 infection.

Salmonella Indiana as a cause of abortion in ewes: Genetic diversity and resistance patterns

Salmonella enterica subspecies enterica Indiana, a food-borne serovar uncommon in most countries, was responsible for an outbreak of abortion in a flock of Lacaune dairy ewes in southern Spain. Drinking water and feedstuff samples were analysed in an attempt to determine the source of the infection. Pigeons (Columba livia) and turtledoves (Streptopelia turtur) in close contact with the ewes were captured and examined for the bacterium. Seventeen S. Indiana strains were isolated from the ewes and wild birds and the genetic similarity among them analysed by Pulsed Field Gel Electrophoresis (PFGE) after the digestion of their genomic DNA with the restriction enzyme XbaI. The results suggest the wild birds might be responsible for the outbreak in the ewes. The strains recovered were fully susceptible to 15 out of the 16 antimicrobial agents tested: ampicillin, amoxycillin clavulanate, cephalothin, ceftriaxone, gentamicin, neomycin, streptomycin, tetracycline, ciprofloxacin, enrofloxacin, sulphonamides, trimethoprim-sulphamethoxazole, apramycin, colistin and chloramphenicol. Differences in the resistance pattern to nalidixic acid were observed; 11 strains (64.7%) were nalidixic acid resistant (R-Nx) and 6 (35.3%) sensitive (S-Nx). Among the R-Nx strains, a substitution of Gly to Cys at position 81 (Gly81àCys) of the gyrA gene in 10 strains isolated from wild birds and ovine foetuses, and of Asp to Tyr at position 87 (Asp87àTyr) in one strain isolated from ewe faeces, were revealed by sequencing the gene. To control the outbreak, enrofloxacin treatment was administered for 5 days. The same therapy was used to prevent infection during following gestation cycles, administering the antimicrobial agent at presentation and over 4 weeks before birth. Anti-bird meshes and closed drinking and feeding troughs were also installed to prevent further contact of the ewes with wild birds.

Streptococcus suis serotypes associated with different disease conditions in pigs

the yeast in the oral cavity whereas selenium sulphide had no effect (Table 2). Similarly, the frequency of isolation ofM pachydermatis from the oral cavity of the miconazole-chlorhexidine treated dogs (seven of 15) was significantly reduced (P<0.05), whereas selenium sulphide treatment had no such effect (M pachydermatis isolated in 12 of 17 dogs). This study indicates that effective treatment of M pachydermatisassociated seborrhoeic dermatitis with miconazole-chlorhexidine shampoo is not only associated with reductions ofM pachydermatis populations on the skin surface but also in the oral cavity. This might indicate that oral carriage reflects contamination of the mucosae by constant seeding from a reservoir of the yeast on heavily colonised skin, particularly in pruritic dogs. Alternatively, reduced yeast counts in the mouth could have resulted from a direct antifungal effect by transfer of the shampoo to the oral mucosae by licking. Regardless of the mechanism, the reduction in oral cavity populations may be of therapeutic value in reducing transfer of oral populations back to the skin. It is noteworthy that only the more effective of the two shampoos studied had a significant effect on oral carriage. Further studies are required to determine whether there is significant transfer ofM pachydermatis cells between skin and mucosal sites in dogs, and to determine whether treatment of mucosal carriage sites may be of value in the management of recurrent cases of dermatitis in a manner similar to that recently suggested for canine pyoderma (Saijonmaa-Koulumies and others 1998).

Genetic analysis of Streptococcus suis isolates recovered from diseased and healthy carrier pigs at different stages of production on a pig farm.

Streptococcus suis isolates from pigs at different stages of production on a farrow-to-finish farm were characterised by serotyping, pulsed-field gel electrophoresis (PFGE) and production of muramidase-released protein, extracellular factor and suilysin. S. suis was isolated from the tonsils of 81/287 (28.2%) healthy pigs: 16/47 (34%) post-weaning, 18/47 (38.3%) transition, 18/47 (38.3%) fattening and 29/146 (19.9%) sows. A total of 127 S. suis isolates were analysed: 14 from diseased pigs at the post-weaning stage and 113 from the tonsils of healthy pigs. Serotypes 2, 4, 9, 14 and 1/14 were isolated from both diseased and healthy pigs. A total of 83 PFGE profiles were obtained; most isolates (95.2%) were grouped into three clusters (A-C). Animals at different production stages harboured isolates with similar phenotypic and genetic profiles, highlighting the importance of healthy animals in the maintenance of strains responsible for outbreaks of clinical disease.

Distribution of serotypes of Streptococcus suis isolated from diseased pigs in Spain

C. Tarradas, DVM, A. Perea, DVM, C. Borge, PhD, B. Huerta, DVM, I. Luque, DVM, Departamento de Sanidad Animal, Facultad de Veterinaria, Campus Universitario de Rabanales, 14071 C6rdoba, Spain A. I. Vela, DVM, J. Goyache, DVM, L. Dominguez, DVM, J. F. FernAndezGaraizabal, DVM, Departamento de Patologia Animal I (Sanidad Animal), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain Streptococcus suis is well known as a major cause of meningitis, septicaemia, endocarditis, reproductive disorders, pneumonia and arthritis in pigs (Higgins and Gottschalk 1999). It is also considered an important zoonotic agent (Arends and Zanen 1988), able to induce meningitis and septicaemia in human beings, and mainly affects pig handlers (Tarradas and others 2001). At present, 35 different serotypes, based on capsular antigens, are recognised (Higgins and others 1995); serotype 2 is the most frequently isolated capsular type in Italy, France and Denmark, while serotype 9 is more frequently isolated in Germany, the Netherlands and Belgium (Gogolewski and others 1990, Aarestrup and others 1998, Sihvonen and others 1998, Berthelot-Herault and others 2000, Wisselink and others 2000, Allgaier and others 2001). Previous studies carried out in Spain have demonstrated that serotype 2 is the most prevalent serotype associated with different disease conditions in pigs (Prieto and others 1993, Luque and others 1998). This short communication reports on 383 strains of S suis isolated from diseased pigs on 99 farms in Spain between 1998 and 2002 during the course of routine diagnostic procedures. All isolates were grown overnight on Columbia agar (Oxoid) and incubated with nalidixc acid, colistin sulphate (Oxoid) and 5 per cent defibrinated sterile sheep blood at 37°C in aerobic conditions for 18 to 24 hours. Biochemical identification of S suis was performed by conventional tests (Devriese and others 1991, Tarradas and others 1994) and by using the commercial identification system Rapid ID32 Strept (bioMerieux), according to the manufacturers instructions. Capsular typing was carried out by slide agglutination with specific rabbit antiserum against the reference strains of serotypes 1 to 34. Statistical analysis was performed to determine the relationship between S suis capsular types and clinical sources. The chi-squared test was used with sPss 11.0 software. Differences were considered significant when P<0 05. All but four of the 383 S suis isolates (98-9 per cent) could be serotyped using the 34 capsular types, and showed a great serotype diversity, with 15 different capsular types being identified (Table 1). Capsular types 9 (54 per cent) and 2 (19.3 per cent) were the most frequently isolated serotypes, followed by serotypes 7 (6.3 per cent), 8 (5.7 per cent), 3 (4.2 per cent) and 1/14 (3.6 per cent). The other serotypes were isolated at a frequency of 1 per cent or less (Table 1). Serotypes 19, 21 and 23 had not been isolated previously from diseased pigs in Spain. The prevalence of serotype 2 was approximately 50 per cent lower than that usually reported for this capsular type in Spain, while serotype 9 was isolated in a proportion much higher than that previously found (Prieto and others 1993, Luque and others 1998, Tarradas and others 2001). It is known that healthy carrier pigs harbouring the organism in their palatine tonsils are involved in the spread of S suis infection (Clifton-Hadley 1985, Gottschalk and Segura 2000, Berthelot-Herault and others 2001). The high frequency of serotype 9 observed in this study could be related to the increased importation of animals from countries in which this capsular type has a high prevalence, such as Germany, the Netherlands and Belgium (Aarestrup and others 1998, Wisselink and others 2000, Allgaier and others 2001). Recent studies based on the multilocus sequence typing scheme of S suis demonstrated genetic diversity among isolates, suggesting that capsular genes may move horizontally through the S suis population (King and others 2002), which would also explain the changes in the distribution of the serotypes. The isolates were recovered from pigs exhibiting a variety of clinical disease conditions (Table 1). Nervous disorders (52.2 per cent) and septicaemia (32.9 per cent) represented the majority of the cases, while other conditions associated with S suis infection included arthritis (2.9 per cent) and metritis ( 16 per cent). These results are similar to those of previous studies in Spain (Luque and others 1998). Of particular interest is the high frequency (10-4 per cent) of cases of endocarditis, characterised by the production of a valvular endocarditis, which had not been previously described, probably because endocarditis does not usually produce clinical signs in live animals (Pedersen and others 1981, Sanford and Park 1987). The relationships between serotypes and clinical disease conditions are shown in Table 1. A high number of isolates belonging to serotype 9 (68 per cent) were recovered from pigs with nervous disorders, while the septicaemia cases associated with this serotype represented a lower prevalence (19.8 per cent). Serotype 2 was also recovered from pigs with nervous disorders (32.4 per cent) and septicaemia (45.9 per cent). Endocarditis cases were associated with serotypes 2, 3, 7, 8 and 9. The statistical association between serotypes and clinical conditions was only determined for the most frequent serotypes. The majority of the strains of serotype 9 (68.1 per cent) were isolated from pigs with nervous disorders, while 63 6 per cent of the strains of serotype 8 were isolated from pigs with septicaemia. The association between these serotypes and clinical source was statistically significant (P<0.05). There was no significant association between strains of serotype 2 and 7 and the clinical source. In conclusion, this study shows that a varied distribution of S suis serotypes has caused a variety of infections in pigs over the past few years in Spain. Serotype 9 has emerged as the predominant serotype and new serotypes ( 19, 21 and 23) have been isolated from diseased pigs in the country. This pathogen remains the main cause of nervous disorders and