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Streptococcus suis(S. suis) is an important zoonotic pathogen that apart from causing huge economic losses to the global swine industry, can also cause severe illness and even death in humans. Antibiotics are commonly used to treat and controlS. suisinfections, contributing to the increasing prevalence of antimicrobial-resistantS. suis, which has attracted the attention of the global health community (1-3). Therefore, monitoring the prevalence of antimicrobial resistance-related genes inS. suisand studying its antimicrobial resistance characteristics will provide guidance for the clinical treatment ofS. suisinfections. In this study,S. suisisolates from 20 provincial-level administrative divisions (PLADs) in China were collected and analyzed from 2011 to 2019, together with the availableS. suissequencing data (with detailed host information) in the National Center for Biotechnology Information (NCBI) database, and a global surveillance ofS. suisresistance was carried out according to several epidemiological dimensions. The results demonstrated that the prevalence of antimicrobial resistance genes inS. suiswas generally higher in China than in other countries and that the prevalence was generally higher in the pig isolates compared to the human isolates. The prevalence of resistance genes varied with serotype; however,erm(B), a gene mediating macrolide resistance, andtet(O), a gene mediating tetracycline resistance, were both prevalent across different serotypes. The results of drug sensitivity experiments indicated a high and concerning level of antimicrobial resistance in theS. suisisolated in China. Therefore, public health practitioners should pay attention to the rational use of medicines, reduce the abuse of therapeutic drugs, and promote the scientific use of medicines in farming to protect the lives of animals and humans.
In this study, nasal swabs, lung, spleen, liver, and lymph nodes of healthy and diseased pigs from 20 PLADs across China were collected and analyzed. The analysis data were then combined with theS. suissequencing data (with detailed host information) in the NCBI database. The samples were collected strictly according to the relevant standard protocols, as all the samples were collected using Eswab tubes containing culture media and then stored and transported appropriately to the handling laboratories using ice packs.S. suisisolation was performed as described in previous studies (4). Genomic DNA ofS. suiswas extracted using the Wizard Genomic DNA Purification Kit (Promega, Beijing, China) following the manufacturer’s instructions. DNA libraries were constructed using the KAPA HyperPrep Kit Illumina platforms (Roche, Basel, Switzerland) following standard protocols and then sequenced on an Illumina HiSeq X Ten platform (Annoroad, Beijing, China). Illumina sequencing reads for each isolate were processed using Trimmomatic with an average quality cutoff of 20 (2.3 million average reads per sample). Finally, the high-quality reads were assembled as contigs of at least 500 bp with SPAdes (with parameter–careful) (version 3.13.1, St. Petersburg Academic University,
https://github.com/ablab/spades ). via the Unicycler (version 0.4.7, The University of Melbourne,https://github.com/rrwick/Unicycler ) assembly pipeline (5). The quality of the genome sequences was checked using the default settings in QUAST (version 5.0.2, St. Petersburg Academic University,https://github.com/ablab/quast ) (6). Identification ofS. suisserotypes was performed using the protocol described by Athey et al. (7). Resistance genes were identified using the SRST2 Toolkit (version 0.2.0, The University of Melbourne,http://katholt.github.io/srst2/ ). Minimum inhibitory concentrations were determined by the broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute guidelines (CLSI, 2015: M100-S25).During 2011–2019, a total of 436S. suisisolates were collected from 20 PLADs in China, including 271 isolates from healthy pigs and 165 from diseased pigs (Table 1). Upon analysis, all 436 isolates were identified asS. suis. Genomic analysis revealed 17–1,753 contigs per isolate, with genome sizes ranging from 1,949,027 to 2,642,630 bp and an average GC content of 41.12%. Additionally, 934 sequencedS. suisisolates with specific host sources were screened from 2,452 isolates in the NCBI database, including 461 isolates derived from humans, 129 from healthy pigs, and 344 from diseased pigs. Altogether, 1,370S. suisisolates were obtained from China, Vietnam, Thailand, the Netherlands, the United Kingdom and Japan, and majority of them were obtained during 2011-2019. A total of 27 distinct serotypes were identified from the 1,370 isolates, with the predominant serotype being serotype 2 (n=686), which accounted for 50.1% of the isolates, followed by serotype 3 (n=75, 5.5%). Furthermore, 57 (4.2%), 54 (4.0%), 50 (3.6%), 32 (2.3%), 32 (2.3%), and 29 isolates (2.1%) were identified as belonging to serotype 9, 7, 4, 1, 8, and 14, respectively.
The predicted antimicrobial resistance genes using the SRST2 Toolkit (http://katholt.github.io/srst2/) in all 1,370S. suisisolates indicated that the prevalence and number of antimicrobial resistance genes were diverse among the different serotypes (Figure 1). Among all the resistance genes,erm(B) andtet(O) were highly observed in all serotypes (Figure 1A). The newly characterizedsrpAgene, which confers resistance to streptogramin A, pleuromutilins, and lincosamides, was found in 60 isolates (4.4%), mainly in serotypes 2, 3, 4, 9, 12, 21, 25, 29, 30, and 31 (dominant serotype 2) (8). Further, the prevalence of antimicrobial resistance genes in the isolates from China was generally higher than that in the isolates from other countries. The prevalence of antimicrobial resistance genes in human isolates was lower than that in pig isolates (Figure 1C). Drug sensitivity results showed that the antimicrobial resistance rate ofS. suiswas the highest for macrolides (91.5%) and the lowest for β-lactams (3.9%–15.4%) (Table 2), consistent with the genomic analysis results of antimicrobial resistance.
Figure 1.Prevalence and number of resistance genes among different serotypes in 1,370Streptococcus suisisolates from 6 countries. (A) Frequencies of AMR genetic determinants within different serotypes, calculated across 22 serotypes. (B) Comparison of the number of AMR genes among the different serotypes. The median is represented by a black line. (C) Comparison of AMR gene prevalence among different countries or different hosts. The horizontal axis represents antimicrobial resistant genes and the vertical axis represents countries or host sources.
Note: In Figure 1A, cells indicate absence (white) or presence (colored by proportion) of each AMR determinant. The horizontal axis represents 54 antimicrobial resistance genes of 13 antibiotic classes, and the vertical axis represents 22 serotypes and none-type (NT), the number of which is greater than two. In Figure 1B, the median is represented by a black line. In Figure 1C, the horizontal axis represents antimicrobial resistant genes and the vertical axis represents countries or host sources.Abbreviations: CN=China; OT=Other countries; H=Human; HP=Healthy pig; DP=Diseased pig; AMR=Antimicrobial resistance; ARG=Antibiotic resistance gene.PLADs Host source (N) Healthy pig Diseased pig Anhui 12 18 Beijing 27 0 Chongqing 28 0 Guangdong 57 0 Guizhou 1 0 Hebei 1 0 Heilongjiang 8 0 Henan 25 52 Hubei 18 22 Hunan 16 16 Inner Mongolia 1 0 Jiangsu 2 0 Jiangxi 2 19 Ningxia 1 0 Qinghai 2 0 shaanxi 0 18 Shandong 19 0 Shanxi 0 20 Sichuan 43 0 Xinjiang 8 0 Total 271 165 Note: Sequencing data obtained from the database were not shown.
Abbreviations: PLADs=provincial-level administrative divisions; N=number.Table 1.Characteristics and prevalence of 436Streptococcus suisfrom the 20 PLADs, China, 2011–2019.
Antimicrobials MIC values (μg/mL) Drug resistance rate (%) 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1,024 Aminoglycosidesa 2 0 0 5 15 28 68 30 18 25 15 41 51 8 58.8% Lincosamideb 9 0 0 0 69 0 0 1 9 7 12 75 106 18 74.5% Fluoroquinolonesc 100 25 24 73 2 24 16 23 13 6 0 0 0 0 27.5% Tetracyclinesd 0 6 0 2 9 45 32 33 25 39 43 5 1 0 58.2% Macrolidese 2 4 20 0 3 19 22 21 19 17 51 111 17 0 91.5% Chloramphenicolf 0 6 3 42 64 77 25 23 36 19 9 2 0 0 37.3% Cephalosporinsg 163 24 26 11 6 64 9 2 1 0 0 0 0 0 3.9% Penicillinsh 83 28 16 32 100 26 10 4 7 0 0 0 0 0 15.4% Note: Refer to the streptococcal drug sensitive threshold.
Drug resistance threshold (μg/mL): a≥16, b≥4, c≥2, d≥8, e≥1, f≥8, g≥8, h≥4.
Abbreviation: MIC=minimum inhibitory concentration.Table 2.The MIC distribution of 306Streptococcus suisisolated in China (n=306).
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In this study, the prevalence of antimicrobial resistance genes inS. suiswas found to differ among the isolates from different sources; the prevalence of these genes inS. suiswas generally higher in China than in foreign countries, which may be related to suboptimal drug use in breeding farms in China, indicating that the antimicrobial resistance ofS. suisin China is a serious public health problem. With respect to the host source, the prevalence ofS. suisantimicrobial resistance genes in the human isolates was lower than that in the pig isolates. This may be because most human isolates were serotype 2 and the prevalence of antimicrobial resistance genes in serotype 2 was generally lower than that in other serotypes, though the serotype 2 isolates accounted for a large proportion of the population. In general, the prevalence oferm(B), which mediates macrolide resistance, andtet(O), which mediates tetracycline resistance, was high among the isolates from all sources; however, the prevalence of these two genes in serotype 2 was lower than that in other serotypes, although serotype 2 represents the dominant serotype of pathogenicS. suis. Tetracycline resistance in porcine streptococci has become a major problem worldwide and is closely related to the widespread use of tetracyclines in the pig industry. The antimicrobial resistance gene prevalence and the antimicrobial resistance phenotype ofS. suisevaluated in this study indicate the problem of the antimicrobial resistance ofS. suisin China to be prominent, and timely actions should be taken to avert an imminent crisis. The results further provide important guidance and reference values for clinical drug use.
The findings of this study were consistent with those of some previous studies. Hout et al. showed that among the 1,163 isolates ofS. suiscollected in the Netherlands during 2013–2015, 78.4% were resistant to tetracycline and 48.1% to clindamycin, with the resistance rate for β-lactams being less than 5% (9). Another study by Zhang et al. investigating the drug sensitivity of 421S. suisisolates from China found that the antimicrobial resistance rates for tetracycline, macrolides, and sulfonamides were more than 60%, and the antimicrobial resistance rate for β-lactams was only 9.5% (10). Based on these results, β-lactam antibiotics can still be used as the drugs of choice forS. suisinfections.
This study was subject to some limitations. While the isolates used in this study for genomic analysis covered most parts of China; the geographical locations of the isolates whose data was sourced from GenBank were not evenly distributed. In addition, most of the isolates from Vietnam belonged to serotype 2.
In summary, data of 1,370 isolates ofS. suisfrom 6 countries were investigated for antimicrobial resistance and classified according to different characteristics for comparative analysis. The prevalence of macrolide and tetracycline resistance genes was the highest. The prevalence of the resistance genes varied with the source; it was lower in the human isolates ofS. suisthan in the pig isolates. Based on these findings, to limit the extensive spread of antimicrobial-resistantS. suis, a series of interventional policies are needed to address the impending antimicrobial resistance crisis and to develop appropriate dosing strategies for the different strains ofS. suiswith different hosts to protect the health of humans and animals under the “One Health” approach.
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Research staff at College of Veterinary Medicine, China Agricultural University.
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No conflicts of interest.
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