Hilde E. Smith

Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries

Streptococcus suis strains (n=411), isolated from diseased pigs in seven European countries were serotyped using specific antisera against serotype 1 to 28, and were phenotyped on the basis of their muramidase-released-protein (MRP) and extracellular-factor protein (EF) production. Overall, S. suis serotype 2 appeared to be most prevalent (32%), followed by serotype 9 (20%) and serotype 1 (12%). Serotype 2 was most frequently isolated in France, Italy and Spain, whereas serotype 9 was most frequently isolated in Belgium, The Netherlands and Germany. In the United Kingdom serotypes 1 and 14 were most frequently isolated. High percentages of S. suis serotype 1, 2, 1/2 and 14 strains, isolated from tissues associated with S. suis infections such as brain, serosa, joint, heart and organs expressed the EF-protein, indicating that in these serotypes expression of EF is likely to be associated with virulence. In contrast, strains belonging to serotype 7 and 9, isolated from tissues associated with S. suis infections did not produce EF. These results strongly suggest that in the serotypes 7 and 9 EF expression is not related to virulence. More than 80% of the S. suis serotype 9 strains produced an MRP* protein, a high molecular variant of the 136kDa MRP. Expression of MRP* in serotype 9 strains is possibly associated with virulence.

Rapid Evolution of Virulence and Drug Resistance in the Emerging Zoonotic Pathogen Streptococcus suis

Background Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. Methodology/Principal Findings The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, ∼40% of the ∼2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three ∼90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. Conclusions/Significance The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.

Contribution of Fibronectin-Binding Protein to Pathogenesis of Streptococcus suis Serotype 2

ABSTRACT In the present study we investigated the role of the fibronectin (FN)- and fibrinogen (FGN)-binding protein (FBPS) in the pathogenesis of Streptococcus suis serotype 2 in piglets. The complete gene encoding FBPS from S. suis serotype 2 was cloned in Escherichia coli and sequenced. The occurrence of the gene in various serotypes was analyzed by hybridization studies. The FBPS protein was expressed in E. coli and purified, and binding to human FN and FGN was demonstrated. The induction of antibodies in piglets was studied upon infection. An isogenic mutant unable to produce FBPS was constructed, and the levels of virulence of the wild-type and mutant strains were compared in a competitive infection model in young piglets. Organ cultures showed that FBPS was not required for colonization of the tonsils but that FBPS played a role in the colonization of the specific organs involved in an S. suis infection. Therefore, the FBPS mutant was considered as an attenuated mutant.

Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry.

To gain more information on the genetic basis of the rapid increase in the number of isolates exhibiting non-wild type Minimum Inhibitory Concentrations (MICs) for cefotaxime observed since 2003, beta-lactamase genes of 22 Salmonella enterica and 22 Escherichia coli isolates from broilers in 2006 showing this phenotype were characterized by miniaturized micro-array, PCR and DNA-sequencing. Presence and size of plasmids were determined by S1-digest pulsed-field gel electrophoresis and further characterized by PCR-based replicon typing. Transfer of resistance plasmids was tested by conjugation and transformation experiments. To link resistance genes and plasmid type, Southern blot hybridization experiments were conducted. In 42 isolates, five (bla(CTX-M-1), bla(CTX-M-2), bla(TEM-20), bla(TEM-52), bla(SHV-2)) different extended spectrum beta-lactamase (ESBL)-genes and two (bla(ACC-1), bla(CMY-2)) AmpC-genes were present. Three of the detected ESBL-genes (bla(CTX-M-1), bla(TEM-52) and bla(CTX-M-2)) were located on similar types of plasmids (IncI1 and IncHI2/P) in both E. coli and Salmonella. Two other detected ESBL- and AmpC-genes bla(SHV-2) and bla(CMY-2) respectively (on IncK plasmids), were only found in E. coli, whereas the AmpC-gene bla(ACC-1) (on non-typable plasmids), and the ESBL-gene bla(TEM-20) (on IncI1 plasmids), were only detected in Salmonella. In two isolates, no ESBL- or AmpC-gene could be detected through these methods. The increase in the number of E. coli and S. enterica isolates from the gastro-intestinal tract of broilers exhibiting non-wild type MICs for cefotaxime is mainly due to an increase in IncI1 plasmids containing bla(CTX-M-1). The reason for the successful spread of this plasmid type in these species is not yet understood.

Extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers

OBJECTIVES The aim of this study was to establish the prevalence of extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase-producing Escherichia coli at Dutch broiler farms and in farmers and to compare ESBL/AmpC-producing isolates from farmers and their animals. METHODS Twenty-five to 41 cloacal swabs collected from broilers at each of 26 farms and 18 faecal samples from 18 broiler farmers were analysed for determination of the presence of ESBL/AmpC-producing E. coli. ESBL/AmpC genes were characterized by microarray, PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing or restriction fragment length polymorphism. E. coli genotypes were determined by multilocus sequence typing. RESULTS Birds from all farms were positive for ESBL/AmpC-producing E. coli, and on 22/26 farms the within-farm prevalence was ≥ 80%. Six of 18 farmers carried isolates containing ESBL/AmpC genes bla(CTX-M-1), bla(CMY-2) and/or bla(SHV-12), which were also present in the samples from their animals. In five of these isolates, the genes were located on identical plasmid families [IncI1 (n = 3), IncK (n = 1) or IncN (n = 1)], and in isolates from two farmers the genes were carried on identical plasmid subtypes (IncI1 ST12 and IncN ST1, where ST stands for sequence type) as in the isolates from their animals. CONCLUSIONS This study shows a high prevalence of birds carrying ESBL/AmpC-producing E. coli at Dutch broiler farms and a high prevalence of ESBL/AmpC-producing E. coli in farmers. This is undesirable due to the risk this poses to human health. Future research should focus on identification of the source of these isolates in the broiler production chain to make interventions resulting in reduction of these isolates possible.

Relatedness of Streptococcus suis isolates of various serotypes and clinical backgrounds as evaluated by macrorestriction analysis and expression of potential virulence traits.

ABSTRACT We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains.

Identification and Characterization of Two Temperature-Induced Surface-Associated Proteins of Streptococcus suis with High Homologies to Members of the Arginine Deiminase System of Streptococcus pyogenes

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.

Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis

Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10ΔccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis.

Presence of ESBL/AmpC -Producing Escherichia coli in the Broiler Production Pyramid: A Descriptive Study

Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one−/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0–24% to 96–100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms.

Multiplex PCR Assays for Simultaneous Detection of Six Major Serotypes and Two Virulence-Associated Phenotypes of Streptococcus suis in Tonsillar Specimens from Pigs

ABSTRACT Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2- (and 1/2-) specific cps gene and the epf gene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on the luc gene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected with S. suis strains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection of S. suis strains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness for S. suis serotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.