Streptococcus suis infection is considered a major problem worldwide in the swine industry, resulting in great economic losses.1 Pigs may acquire S suis via both vertical and horizontal transmission. Colonized animals typically harbor the organism in their tonsils and may never develop disease (carrier animals). On the contrary, some carrier piglets eventually develop bacteremia, septicemia, meningitis, or all three, due to dissemination of S suis from tonsils or other mucosal surfaces or both, usually when maternal antibodies decline.2 Of the various manifestations of the disease, septicemia and meningitis are by far the most striking, but endocarditis, pneumonia, and arthritis may also be observed. Nevertheless, in hyperacute cases of infection, pigs are often found dead with no premonitory signs of disease.3 Moreover, S suis has zoonotic potential, causing septicemia with or without septic shock, meningitis, and other less common infections in humans.4 Human infection with S suis has become a serious zoonosis and has been observed in many countries where intensive swine production is practised.2,5 Overall, three human cases of S suis meningitis have been reported in Italy, one in the 1990s and two in 2007. One of the two cases reported in 2007 was identified in the geographical area covered by this study and was characterized as S suis serotype 2.6,7 A total of 35 serotypes of S suis have been described and defined on the basis of the antigenicity of their capsular polysaccharide (CPS).8 Streptococcus suis strains differ in virulence, and strains of the same serotype can be differentiated by the expression of virulence-associated factors, including muramidase-released protein (MRP, encoded by mrp); a peptidoglycan-associated protein probably acting as an adhesin; an extracellular protein factor (EF, encoded by epf); and suilysin (SLY, encoded by sly), which is a thiol-activated hemolysin with a cytotoxic effect that might allow penetration into deeper tissues.9 Muramidase-released protein, EF, and SLY have been considered the major virulence-associated markers of S suis 2,10 and the arginine deiminase enzyme (ADS, encoded by arcA) has been recently described11 and seems to play an important role in survival of the bacterium by increasing resistance to acidity. The gene cps2 is specific for S suis serotypes 2 and 1/2 and is considered a fifth virulence factor.12 The virulence factors MRP, EF, and SLY are associated with S suis serotype 2 strains in Europe and Asia, but not with the less virulent North American strains.12 The efficacy of many commercially available S suis killed whole-cell vaccines is poor because protection is limited to homologous strains.13 Previous studies revealed a wide diversity of antimicrobial resistance and varied distribution of virulence-associated factors in different serotypes of S suis, but few studies have focused on the relationship between antimicrobial resistance and virulence factors.14-17 To our knowledge, this study represents the first characterization of clinical strains of S suis isolated from dead piglets from farms located in southern Italy (Sardinia) between 2012 and 2014. Hence, the objective of this work was to determine the antimicrobial resistance, serotypes, and virulence factors of these clinical isolates in order to estimate a correlation among these three characteristics.

Materials and methods

This study did not require ethical review because the activities comprised a part of a periodic, routine diagnostic monitoring program and did not involve animal experimentation.

Bacterial strains and culture conditions

In this study, 39 strains of S suis were recovered from pigs at necropsy at the Istituto Zooprofilattico Sperimentale della Sardegna “G. Pegreffi” (Public Veterinary Diagnostic Laboratory, Sardinia, Italy) between 2012 and 2014 from samples submitted for disease diagnosis in piglets from farms located in southern Italy (Sardinia). The samples were collected from a variety of tissues from dead pigs with lesions such as pneumonia or pleurisy or both, meningitis, endocarditis, and septicemia. Streptococcus suis strains were isolated frequently from pigs between 5 and 10 weeks of age. The bacterial strains were identified as S suis by cultural methods. Specimens were inoculated onto Columbia agar plates (Oxoid Ltd, Basingstoke, United Kingdom) supplemented with 5% sheep blood and incubated in aerobic conditions at 37°C for 24 to 48 hours. Two to three colonies that did not exceed 1 mm in diameter and exhibited α-hemolysis were picked from each plate to subculture on Columbia 5% blood agar plates and were incubated in the same manner. Suspicious colonies, ie, gram-positive cocci negative on the catalase test, were confirmed by the API-20STREP system (BioMérieux, Marcy l’Etoile, France).

DNA extraction and PCR conditions

Genomic DNA was isolated and purified with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) using the method for gram-positive microorganisms indicated in the manufacturer’s instructions. Genomic DNA was stored at -20°C until further processing. Multiplex PCR (mPCR)18 was used to determine the presence of cps2, epf, sly, mrp, and arcA genes. In order to evaluate the sensitivity of the mPCR assays, S suis type strain DSMZ 9682 was used as a reference strain. The sequences of oligonucleotidic primers are listed in Table 1. The mPCR mixture contained PCR buffer 1X (Invitrogen, Cergy Pontoise, France), 2 mM MgCl2 (Invitrogen); a 300-μM concentration of each of deoxynucleoside triphosphate (Invitrogen), 0.1 μM of each primer for epf, 0.06 μM of each primer for cps2, 0.03 μM of each primer for sly, 0.05 μM of each primer for mrp, and 0.06 μM of each primer for arcA; 0.04 U of Taq DNA polymerase (Invitrogen); and 50  ng of DNA template. UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Waltham, Massachusetts) was used as negative control. The reaction procedure consisted of an initial denaturation at 94°C for 2 minutes, 40 cycles at 94°C for 60 seconds, 55°C for 60 seconds, and 72°C for 90 seconds, with a final extension at 72°C for 5 minutes. The amplified products were separated in a 2% agarose gel in Tris-acetate-EDTA (TAE) buffer (Thermo Fisher Scientific) for 1 hour at a constant voltage of 125 V. Amplified products were stained with Syber Safe (Thermo Fisher Scientific) and detected by ultraviolet transillumination. The 100 bp Smart Ladder (Invitrogen) was used as a molecular size standard.

Antimicrobial susceptibility testing

The 36 strains determined to be S suis cps2+ were tested for susceptibility to nine antimicrobials according to the Clinical and Laboratory Standards Institute (CLSI) 2013 guidelines.19 Three to four colonies from an overnight culture on Columbia agar supplemented with 5% sheep blood were suspended in Mueller Hinton (MH) broth (Becton Dickinson, Pont de Claix, France). The suspension was adjusted to a 0.5 McFarland standard and diluted to obtain an inoculum of 106 colony-forming units (CFU) per mL of S suis. For each isolate, two plates were inoculated by flooding MH agar supplemented with 5% sheep blood with the diluted suspension (4 mm depth) (Becton Dickinson). The antibiotic discs were placed with a disc dispenser (Bio-Rad, Hercules, California), and plates were incubated at 37°C for 18 hours. Antimicrobial agents tested were as follows: amoxicillin (10 μg per disc), amoxicillin-clavulanic acid (30 μg per disc), penicillin G (10 units per disc), ampicillin (10 μg per disc), ceftiofur (30 μg per disc), enrofloxacin (5 μg per disc), tetracycline (30 μg per disc), trimethoprim-sulfamethoxazole (25 μg per disc) (Oxoid Ltd), and erythromycin (15 μg per disc) (Cefar Diagnóstica Ltda, São Paulo, Brazil). Escherichia coli ATCC 25922, E coli ATCC 35218, and Enterococcus faecalis ATCC 29212 were used as control strains. The zone of growth inhibition was interpreted as sensitive, intermediate, or resistant. The inhibition zone diameters of S suis strains and the reference strains were measured on the same day using a sliding caliper. For amoxicillin, amoxicillin-clavulanic acid, ceftiofur, erythromycin, penicillin G, and trimethoprim-sulfamethoxazole, disc diffusion susceptibility was tested according to the Clinical and Laboratory Standards Institute (Approved Standard VET01-A4; CLSI, 2013) guidelines specific for S suis strains.19 For ampicillin, enrofloxacin, and tetracycline, specific breakpoints for S suis are not available in CLSI guidelines. For those antimicrobials, interpretation of disc diffusion susceptibility was evaluated according to breakpoints indicated by the manufacturer as specific for Streptococcus species (Oxoid Ltd).

Statistical analysis

The correlation between antimicrobial resistance and virulence factors was studied. Microbial resistance values were expressed as percentages and compared using a Fisher’s exact test. Statistical analysis was performed with SPSS software (version 15.0; SPSS Inc, Chicago, Illinois), and statistical significance was defined at P < .05.

Results

All isolates were determined to be S suis serotype 2 by biochemical characteristics (API -20 strep, BioMerieux SA France) and further confirmed by positive PCR for the genes coding for the 16S rRNA of S suis and for the capsule of S suis serotype 2 (cps2+).

In this study, 39 S suis strains were isolated, and 36 of them (92.3%) belonged to genotype 2 (cps2+). Among the 39 cases, 38.5% were categorized as lung infections, 18.0% as meningitis, 18.0% as endocarditis, and 25.5% as septicemia (cases where S suis was isolated from multiple organs or from mediastinal lymph nodes, spleen, liver, and kidney were classified as septicemia) (Table 2). Numbers of isolates from various organs are shown in Table 3. Virulence factor gene epf was detected in 18.0% of the isolates, whereas virulence factor genes sly, mrp, and arcA were detected in 100% of the isolates. Three genotypes, cps2+/epf+/sly+/mrp+/arcA+, cps2+/epf-/sly+/mrp+/arcA+, and cps2-/epf-/sly+/mrp+/arcA+ were identified, representing 18.0%, 74.6%, and 7.4%, of the isolates respectively (Table 3).

The collection of 36 S suis cps2+ strains was tested for susceptibility to nine antimicrobials (Table 4). A high frequency of resistance was observed for tetracycline (88.9%), erythromycin (38.9%), trimethoprim-sulfamethoxazole (16.7%), and enrofloxacin (11.1%). Multiple antimicrobial resistance (two or more antimicrobials) was observed in 58.3% of the S suis isolates. Sensitivity testing showed that ampicillin had the greatest antimicrobial effect on the 36 isolates (100% of strains were susceptible), followed by amoxicillin, amoxicillin-clavulanic acid, penicillin G, and ceftiofur (Table 4).

The correlation between antimicrobial resistance and virulence factors epf+/epf- was evaluated (Table 5). The virulence factors cps2+/cps2- were not analyzed for any correlation with antimicrobial resistance because of the small number of cps2- strains. (Table 2). Since no strains resistant to ampicillin were found, no correlation data were collected for this antibiotic. The virulence factor epf was not correlated with resistance to multiple antibiotics in this study.

Discussion

Streptococcus suis is a major swine pathogen, responsible for important economic losses to the porcine industry worldwide. In western countries, S suis infections in humans have mostly been restricted to workers in close contact with pigs or swine by-products. However, in Southeast and East Asia, this bacterium also affects the general population and thus represents a significant public-health concern.2 During the 3 years of monitoring, the organs of all piglets dead within the geographical district of interest were screened for S suis infection. Streptococcus suis strains isolated in this study represent the entire set of clinical strains whose infection led to piglet deaths. The number of isolates was small because this study focused attention on strains that cause piglet mortality. Strains were isolated from different farms; nevertheless, clonality among isolates cannot be excluded. The small number of studied strains and the potential for clonality can add bias to the evaluation of correlation between antimicrobial resistance and virulence factors.

An accurate isolation strategy was carried out in order to avoid selective isolation of clonal strains from the same farm. Different capsular serotypes display various clinical manifestations and differ vastly among countries. The most prevalent capsular gene in the isolates in this study was cps2; cps2+ strains are known to be highly virulent. In this study, cps2- strains caused endocarditis and septicemia. This is not surprising, because other cps2- serotypes in Europe, particularly with the profile cps2-/epf-/sly+/mrp+/arcA+, have proved to be highly virulent and can cause septicemia and meningitis in pigs.20 We found that mrp+/sly+/epf+ and mrp+/sly+/epf- genotypes were predominant among the tested isolates. The arcA gene was identified in all strains, confirming previous studies.21 From European epidemiological studies10 and experimental infections in pigs,22 strains of the sly+/mrp+/epf+ genotype are known to be highly virulent. This type was significantly associated with systemic infection in pigs and was highly pathogenic to mice.17 This genotype has also been identified in many isolates from human clinical cases.20 Though epf is not an essential virulence factor for S suis serotype 2 strains, it is probably associated with other factors that play a more crucial role in determining virulence and host specificity in S suis strains.23

Moreover, in this study, resistance of S suis to antibiotics commonly used in pig farms in the area was examined. A high rate of resistance was observed for tetracycline (88.9%) and erythromycin (38.9%). In this study, S suis had the greatest susceptibility to beta-lactam antibiotics and the greatest resistance to erythromycin and tetracycline. High rates of resistance to macrolides and tetracyclines suggest widespread resistance to these antibiotics in Italy.21 In Europe, rising rates of resistance have been attributed to the intensive use by swine breeders of the macrolide-class antibiotics, tylosin as a growth promoter and tetracycline as a therapeutic agent.20 Co-resistance to macrolides and tetracyclines can be explained by the fact that tetracycline and erythromycin resistance determinants are often linked to mobile genetic elements.24 The trend of S suis resistance to macrolides and tetracyclines has been reported worldwide.25 Studies of genetic resistance traits have demonstrated that erm(B) and mef(A) are involved in macrolide resistance, whereas tet(M) and tet(O) are involved in tetracycline resistance.21 Resistance to erythromycin is a concern for public health, as macrolide drugs are important for therapeutic treatment of severe streptococcal cases in humans.17 Resistance of the isolates in the present study to penicillin and ceftiofur was lower than resistance to other tested antibiotics, but was, however, clinically significant. Thus, development of resistance to these antibiotics would reduce the efficacy of antibiotic treatment. To the best our knowledge, this study represents the first epidemiological investigation of lethal cases of S suis infection in piglets in southern Italy (Sardinia). Moreover , this study represents a first attempt to correlate antimicrobial resistance and virulence factors in S suis isolated in southern Italy (Sardinia). The results reveal that the majority of S suis isolates from dead pigs carry multiple virulence factors and that cps2+ strains display resistance to multiple antimicrobials. Infection with invasive S suis requires antibiotic treatment. Possession of both antimicrobial resistance and virulence makes these pathogenic strains potentially highly dangerous to both animal production and public health. Worldwide, S suis serotype 2 is the most frequently isolated serotype.

Implications

•  Streptococcus suis strains from piglets from participating farms located in southern Italy (Sardinia) were resistant to tetracycline and erythromycin between 2012 and 2014.

•  Under the conditions of this study, antimicrobial resistance and genomic virulence factors in S suis isolated from swine are not correlated.

Acknowledgements

We thank Dr Timo G. Lutz for his revision of the language of this manuscript.

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use this information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in its country or region.

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