Antimicrobial Resistance,Serotypes,and Virulence Factors of Streptococcus suis Isolates from Diseased Pigs

Abstract

Streptococcus suis isolates from diseased pigs were examined for susceptibility to nine antimicrobials, possession of virulence-associated factors (VFs), and distribution of serotypes. The association between antimicrobial resistance (AMR) and serotypes as well as VFs was subsequently assessed. Among the isolates investigated, serotype 2 (66.04%) was mostly prevalent, followed by serotypes 1 (23.27%), 9 (1.26%), and 7 (0.63%), whereas 14 isolates were untypable by the polymerase chain reaction typing method used. Analysis with pulsed-field gel electrophoresis revealed the isolates had diverse DNA macrorestriction patterns. The frequency of antimicrobial resistance among the S. suis isolates was higher than that reported from other countries. It is notable that multiple antimicrobial resistance (three or more antimicrobials) was observed with 98.73% of the S. suis isolates, and the dominant resistance phenotype was erythromycin-tilmicosin-clindamycin-chloramphenicol-levofloxacin-ceftiofur-kanamycin-tetracycline-penicillin (35.85%). The most prevalent VFs were those encoded by muramidase-released protein (61.64%), followed by suilysin (56.60%) and extracellular factor (46.54%). Presence of VFs and the possession of certain AMR phenotypes were significantly associated as determined by statistical analysis. Together, these findings indicate that the clinical S. suis isolates obtained from diseased pigs in China are genetically diverse, are resistant to multiple antibiotics of clinical importance, and carry known virulence factors.

Introduction

S treptococcus suis, a well-recognized swine pathogen, causes significant clinical problems in countries with intensive swine production. It is also a zoonotic pathogen, posing a threat to public health. The infections by S. suis are associated with different clinical conditions, such as encephalitis, meningitis, arthritis, septicemia, abortion, and endocarditis in swine (Higgins et al., 1992) and septicemia and meningitis in humans (Yu et al., 2006). It is notable that several large human outbreaks of S. suis have been recently described in China (Tang et al., 2006; Ye et al., 2006), suggesting the enhanced zoonotic transmission and virulence of S. suis to humans.

To date, 33 serotypes of S. suis have been described (Hill et al., 2005), and not all serotypes have similar clinical relevance or are equally important in different countries. Serotypes 1, 2, 7, 9, and 14 are most frequently isolated from diseased pigs in Europe (Wisselink et al., 2000), whereas in North America, serotypes 3 and 8 are frequently isolated from diseased animals (Fittipaldi et al., 2009).

Vaccination of piglets against S. suis is hampered by the lack of vaccines protecting against more than one serotype, and the treatment with antimicrobials are hampered by increasing antimicrobial resistance (AMR) in S. suis. To understand and control AMR, an important first step is to establish a surveillance program of AMR. However, this program has not been well developed in China (Zhang et al., 2008).

S. suis isolates differ in virulence, and strains of the same serotype can be differentiated by expression of virulence-associated factors (VFs), such as muramidase-released protein (MRP), extracellular factor (EF), and suilysin (SLY), encoded by mrp, ef, and sly, respectively. Although there was limited evidence confirming that these three factors (those encoded by mrp, sly, and ef) play a critical role in the virulence of S. suis, a positive correlation of these factors with the virulence was seen in European and Asian strains (Gottschalk et al., 2007).

Previous studies showed a wide diversity of AMR and varied distribution of VFs in different serotypes of S. suis (Aarestrup et al., 1998a; Marie et al., 2002; Wei et al., 2009), but there have been few studies that have focused on the relationship between AMR and VFs in S. suis (Marie et al., 2002). Additionally, up to now, such studies have not been reported in China, where S. suis is a significant threat to both animal health and public health. Hence, the objective of this study was to determine the prevalence of serotypes, AMR profiles, and VFs in S. suis isolated from diseased pigs and to estimate the association among AMR, serotypes, and VFs using statistical methods.

Materials and Methods

Bacterial isolates

Large numbers of diseased or dead animals were submitted to the animal hospital of Foshan University for diagnostic purpose in southern China. In total, 159 non-duplicate S. suis isolates were isolated from diseased pigs between February 2008 and October 2010. These diseased pigs were from 60 farms all over Guangdong Province, China. One to three samples were collected from each farm, and each sample was from an individual animal. The collected samples were collected from a variety of tissues, including lung, spleen, brain, heart, joints, and liver. The bacterial strains were identified as S. suis by a specific polymerase chain reaction (PCR) assay (Marois et al., 2004) and confirmed by the API-20E system (bioMérieux, Marcy l'Etoile, France).

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed on the 159 S. suis isolates using a broth microdilution method according to the standardized method described by the CLSI (2009). The antimicrobial agents tested and breakpoints for clarifying S. suis as resistant were as follows: penicillin, 4 mg/L; ceftiofur, 8 mg/L; kanamycin, 64 mg/L; tetracycline, 8 mg/L; erythromycin, 1 mg/L; tilmicosin, 32 mg/L; clindamycin, 1 mg/L; chloramphenicol, 16 mg/L; and levofloxacin, 8 mg/L. Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 served as the reference strains for quality control in minimal inhibitory concentration (MIC) determinations.

Serotyping and virulence genotyping

All of the S. suis isolates were typed for serotypes (1, 2, 7, and 9) by multiplex PCR as described previously (Wisselink et al., 2002). The detection of VFs, including mrp, sly, and ef, was performed using the gene-specific primers (Silva et al., 2006). The PCR products obtained were sequenced, and the DNA sequences were compared with the GenBank database using BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). Clinical isolates confirmed to harbor these genes by DNA sequencing were used as positive controls for the PCR detection.

Pulsed-field gel electrophoresis analysis

The isolates were typed by pulsed-field gel electrophoresis (PFGE) using SmaI (Berthelot-Herault et al., 2002), and the Salmonella Braenderup H9812 strain was used as the molecular standard. The used PFGE conditions were as follows: agarose concentration, 1%; voltage gradient, 6 V/cm; angle, 120°; and run time, 22.2 h. The DNA band analysis was performed by visual inspection following the criteria described previously (Tenover et al., 1995).

Statistical analysis

Statistical analysis was performed with SPSS software (version 17.0; SPSS Inc., Chicago, IL). The chi-square test was used, and a value of p<0.05 was considered significant in this study.

Results

Serotypes of S. suis isolates

Among the 159 S. suis isolates, most (91.19%) of the isolates belonged to serotypes 1, 2, 7, and 9, whereas 14 isolates were untypable using the PCR assays. Serotype 2 was the most prevalent among the typable 145 isolates and accounted for 66.04% of the isolates, followed by serotypes 1 (23.27%), 9 (1.26%), and 7 (0.63%).

AMR phenotypes

The 159 isolates were tested for susceptibility to nine antimicrobials. The most prevalent phenotypes detected were resistant to clindamycin (98.11%), erythromycin (94.97%), chloramphenicol (88.68%), tilmicosin (84.91%), and tetracycline (83.02%), followed by levofloxacin (67.30%), penicillin (66.04%), kanamycin (61.64%), and ceftiofur (55.97%). The 50% and 90% MIC values of ceftiofur were 8 mg/L and 16 mg/L, respectively (Table 1). In addition, it was observed that 98.74% of the isolates were resistant to at least one antibiotic and that98.73% were multidrug resistant (resistant to at least three different antibiotic classes) (Table 2).

Table 1.

Antibiotic Resistance Phenotype of Streptococcus suis Isolates (n=159)

	No. of isolates for which the MIC (mg/L) was
Antibiotic	0.25	0.5	1	2	4	8	16	32	64	128	256	512	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Resistant isolates (%)	Resistance breakpoint (≥mg/L)
Erythromycin	7	1	1	4	3	8	4	4	8	15	38	66	256	512	151 (94.97)	1
Tilmicosin	9	2	3	1	4	2	4	15	8	14	33	64	256	512	135 (84.91)	32
Clindamycin	1	1	0	4	8	9	13	7	3	6	43	64	256	512	156 (98.11)	1
Chloramphenicl	7	1	2	1	1	1	5	17	17	33	59	15	128	256	141 (88.68)	16
Levofloxacin	29	6	5	11	9	5	17	25	21	15	10	6	16	128	107 (67.30)	8
Ceftiofur	27	5	12	15	11	18	60	1	2	1	1	7	8	16	89 (55.97)	8
Kanamycin	22	1	2	5	3	11	17	18	12	13	38	16	32	256	98 (61.64)	64
Tetracycline	17	0	2	1	3	4	3	9	18	28	44	25	128	512	132 (83.02)	8
Penicillin	27	2	5	9	10	2	3	2	1	1	18	79	256	512	105 (66.04)	4

MIC, minimal inhibitory concentration; MIC₅₀ and MIC₉₀, minimal inhibitory concentration yielding 50% and 90% inihibition, respectively.

Table 2.

Distribution of Serotypes, Antibiotic Resistance Patterns, and Virulence Phenotypes Among Streptococcus suis Isolates

	No. of isolates ^a								No. of isolates ^b
Resistance pattern (no. of isolates)	a	b	c	d	e	f	g	h	1	2	7	9	Other
Ery-Til-Cli-Tet (4)	0	0	0	1	0	0	1	2	2	1	0	0	1
Ery-Til-Cli-Chl (5)	1	1	1	1	0	0	0	1	0	5	0	0	0
Ery-Til-Cli-Chl-Tet (3)	1	0	0	0	0	0	0	2	0	3	0	0	0
Ery-Cli-Chl-Kan-Tet-Pen (3)	2	0	0	0	0	0	0	1	0	3	0	0	0
Ery-Til-Cli-Chl-Kan-Tet (7)	2	0	0	2	1	0	0	2	1	6	0	0	0
Ery-Til-Cli-Chl-Cef-Tet-Pen (4)	2	1	0	0	0	0	0	1	1	3	0	0	0
Ery-Til-Cli-Chl-Lev-Tet-Pen (7)	2	1	0	1	0	1	1	1	4	3	0	0	0
Ery-Til-Cli-Chl-Cef-Kan-Tet-Pen (3)	2	1	0	0	0	0	0	0	0	3	0	0	0
Ery-Til-Cli-Chl-Lev-Cef-Kan-Tet (3)	3	0	0	0	0	0	0	0	0	3	0	0	0
Til-Cli-Chl-Lev-Cef-Kan-Tet-Pen (1)	4	1	0	1	0	0	1	0	0	1	0	0	0
Ery-Til-Cli-Chl-Lev-Kan-Tet-Pen (11)	1	1	2	0	0	2	1	4	3	5	0	0	3
Ery-Til-Cli-Chl-Lev-Cef-Tet-Pen (11)	5	1	0	2	0	0	0	3	3	7	0	1	0
Ery-Til-Cli-Chl-Lev-Cef-Kan-Tet-Pen (57)	15	5	3	8	1	6	1	18	13	35	1	1	7

Because the resistance patterns are diverse in this study, only the major resistance patterns represented by at least three isolates are shown here.

Patterns with respect to expression of the virulence-associated factors muramidase-released protein, suilysin, and extracellular factor, encoded by mrp, ef, and sly, respectively, are as follows: a, mrp+sly+ef+; b, mrp+sly+ef–; c, mrp+sly–ef+; d, mrp+sly–ef–; e, mrp–sly+ef+; f, mrp–sly+ef–; g, mrp–sly–ef+; and h: mrp–sly–ef–.

These isolates were identified as serotypes 1, 2, 7, and 9; other means the isolates were those that were untypable using the polymerase chain reaction assays.

Cef, ceftiofur; Chl, chloramphenicol; Cli, clindamycin; Ery, erythromycin; Kan, kanamycin; Lev, levofloxacin; Pen, penicillin; Tet, tetracycline; Til, tilmicosin.

Prevalence of VFs

For the virulence factors, the mrp, sly, and ef genes were detected in 61.6%, 56.6% and 46.5% of the isolates, respectively. A high percentage (37.11%) of the isolates harbored all three genes (mrp+sly+ef+), whereas the triple negative phenotype mrp–sly–ef– accounted for 24.53% of the isolates. Meanwhile, six other phenotypes existed: mrp+sly+ef–, mrp+sly–ef–, mrp–sly+ef–, mrp–sly–ef+, mrp+sly–ef+, and mrp–sly+ef+, representing 11.32%, 10.07%, 6.92%, 4.40%, 3.77%, and 1.89% of the isolates, respectively.

Distribution of VFs among different serotypes

When each serotype was compared with all other serotypes combined, mrp was significantly associated with serotype 1 (p=0.021), serotype 2 (p=0.001), and untypable isolates (p=0.001), and sly was significantly associated with serotype 2 (p=0.001) and untypable isolates (p=0.005), whereas ef was significantly associated with serotype 2 (p=0.017) and untypable isolates (p=0.011). The most prevalent phenotypes among serotype 1 isolates were mrp+sly+ef+(27.03%) and mrp–sly–ef– (27.03%), whereas the most prevalent phenotype was mrp+sly+ef+(46.67%) among serotype 2 isolates.

Association between AMR and serotypes

When each serotype was compated with all other serotypes combined, a very significant association between resistance to clindamycin and serotype 2 (p=0.015) was found. In addition, significant correlations between resistance to chloramphenicol and serotype 2 (p=0.040) and untypable isolates (p=0.033) were also identified in the study.

Association between AMR and VFs

The three virulence factors were found to be related with resistance to different antimicrobials in this study. The mrp gene was significantly associated only with the resistance to tilmicosin (p=0.005), and sly was significantly correlated with resistance to tilmicosin (p=0.006) and kanamycin (p=0.001), whereas ef was significantly associated with four antimicrobials: tilmicosin (p=0.007), levofloxacin (p=0.049), penicillin (p=0.013), and tetracycline (p=0.021).

PFGE analysis

Of the 159 isolates, 145 were successfully typed by PFGE, and 145 different PFGE profiles were obtained according to the previously described criteria (Tenover et al., 1995), suggesting that the isolates in this study are genetically divergent and are not clonally related. This result also suggests that the prevalence of S. suis in pigs in China is not the result of clonal expansion of a particular genotype, but is likely due to transmission of multiple genotypes.

Discussion

S. suis infection is recognized to be a major threat to swine industries worldwide, resulting in great economic losses (Staats et al., 1997; Gottschalk et al., 2007). In this study, we demonstrated high resistance of S. suis isolates to many antimicrobial agents commonly used in China. Similar to previous reports (Zhang et al., 2008; Princivalli et al., 2009), resistance to erythromycin, tilmicosin, clindamycin, and tetracycline was predominant. This high frequency of resistance might be explained by intensive use of tilmicosin and tetracycline (for therapeutic purpose or growth promotion) in pig production in China, and the resistance to erythromycin is a concern for public health because macrolide drugs are important for therapeutic treatment of severe streptococcal cases in humans.

It is interesting that a high incidence of resistance to chloramphenicol was detected in the present study, despite the fact that chloramphenicol was banned for use in veterinary medicine in China 9 years ago. This high incidence of resistance to chloramphenicol is rare in other countries (Kataoka et al., 2000). Antimicrobial resistance can persist when the antimicrobial selection pressures are removed according to the finding of a previous study (Bischoff et al., 2002), as the selection of resistant bacteria can occur through a variety of mechanisms, which may not always be linked to a use of a specific antibiotic.

The incidences of resistance to levofloxacin, kanamycin, penicillin, and ceftiofur of the isolates in the present study were lower than the incidences of resistance to other tested antibiotics, but were still significant (>55%). It is noteworthy that these antibiotics are commonly used in veterinary medicine, and some of them are the primary drugs to treat the infections by S. suis. Thus, development of resistance to these antibiotics would reduce the efficiency of antibiotic treatment. In addition, multidrug resistance phenotypes of S. suis isolates have been reported worldwide (Kataoka et al., 2000; Vela et al., 2005), and in this study we found that 98.73% of the isolates were resistant to three or more antimicrobials and 35.85% were resistant to nine antimicrobials, indicating that the S. suis isolates in China are resistant to a broad range of antibiotics.

The overall detection frequencies of VFs in our study were lower than the reported prevalence of previous studies (Yu et al., 2006; Padungtod et al., 2010). In addition, according to the previous reports, the phenotypes mrp+sly–ef– and mrp–sly–ef– were the most prevalent in the United States and France (Fittipaldi et al., 2009; Wertheim et al., 2009), whereas the genotypes mrp+sly+ef+ and mrp–sly+ef+ were the most prevalent in China as reported by others (Wei et al., 2009). In this study, we found that mrp+sly+ef+ and mrp–sly–ef– genotypes were predominant among the tested isolates. The variations could be due to the difference in the sources of isolates and suggest that detection of the VFs has a limited use in predicting virulence.

The distribution of capsular serotypes varied with different clinical manifestation and differed vastly among countries. Serotype 2 was most frequently isolated in France (Berthelot-Herault et al., 2000), Canada (Higgins and Gottschalk, 1993), and China (Wei et al., 2009), whereas serotypes 1 and 14 were prevalent in the United Kingdom (Wisselink et al., 2000). The predominance of serotype 2 in our study was already reported by other authors (Padungtod et al., 2010; Ngo et al., 2011), and besides this, serotype 1 was also prevalent in this study, which differed from another report in China (Wei et al., 2009), in which serotype 3 was more common. This discrepancy could be due to regional or time differences, or other selective advantages, which favor the selection of different serotypes of S. suis strains under a given environment as reported previously (Aarestrup et al., 1998a).

Serotype-dependent differences in AMR were reported in previous studies. For example, isolates of serotype 2 were found to be frequently resistant to macrolides, tetracycline, and lincosamides (Cantin et al., 1992; Aarestrup et al., 1998b; Marie et al., 2002), although these results were inconsistent with other reports, in which no correlations were found between antimicrobial susceptibility and a particular capsular serotype (Han et al., 2001). In our study, we found that serotype 2 was resistant to phenicols more frequently than other serotypes, and such association has not been described in S. suis in the past. The reason for the observed difference in serotype-associated susceptibility among isolates from China is not known, but may reflect differences in the ability of different serotypes to acquire resistance or differences in regional distribution of different serotypes.

To our best knowledge, this study represents the first attempt to associate AMR with VFs in S. suis. It is important to mention that the association between the two phenotypes could be dependent on the bacterial population, strain, source, and other important factors, and the association may exist at the statistical and genotypic levels. It is interesting that VFs were found to be associated with resistance to certain antimicrobials at the statistical level in this study, including kanamycin, levofloxacin, penicillin, tilmicosin, and tetracycline, which are used commonly in swine production. It is notable that all of the three VFs were significantly associated with the resistance to tilmicosin. Because these antibiotics have been commonly used in swine, the findings suggest that continual use of these antimicrobials could lead to the selection and spread of pathogenic and antibiotic-resistant S. suis. Thus surveillance of virulence and resistance profiles of S. suis isolates is necessary, as this may help to detect the emergence of especially harmful and antibiotic-resistant strains.

S. suis disease is among the leading cause of streptococcal infections and has caused two reported major outbreaks in humans in China (Yu et al., 2006; Ye et al., 2006). Antimicrobial therapy is one of the important measures to control infections by S. suis and other pathogens in swine production. Thus food-producing animals that harbor antibiotic-resistant S. suis isolates are common, and these animals might serve as a reservoir of antibiotic resistance. The findings of the present study indicated that the use of antimicrobials could lead to the selection and spread of pathogenic and antibiotic-resistant S. suis isolates, and this may pose a great challenge for the public health, as the antibiotic-resistant pathogens are zoonotic and can be transmitted to humans through livestock according to a previous study (Moubareck et al., 2003).

Conclusions

The results in this study reveal that the majority of S. suis isolates from diseased pigs belong to serotypes 1 and 2, carry multiple VFs, and display resistance to multiple antimicrobial compounds. VFs showed significant associations with the resistance to certain antibiotics, suggesting that AMR and VFs are co-selected for S. suis. Because infections with invasive S. suis require antibiotic treatment, possession of both antimicrobial resistance and virulence makes these pathogenic strains potentially more dangerous to both animal production and public health. To our best knowledge, this is the first report of the association between AMR and VFs at the statistical levels in S. suis.

Footnotes

Acknowledgments

The authors are grateful to Professor Qijing Zhang (Iowa State University, Ames) for critically reviewing the manuscript. This work was supported by the National Science Fund for Distinguished Young Scholars (grant 31125026), the Special Fund for Agro-scientific Research in the Public Interest (grant 201203040), and the National Natural Science Foundation of China (grant U0631006).

Disclosure Statement

No competing financial interests exist.

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