• View in gallery

    Trend of florfenicol resistance of invasive non-typhoidal Salmonella strains from humans in China (2007–2016). Denominator in brackets indicate the total number of blood culture–positive non-typhoidal Salmonella strains cultured in each year. The numerator in brackets indicates the florfenicol-resistant strains.

  • View in gallery

    XbaI pulsed-field gel electrophoresis profiles and detection of resistance genes of 34 florfenicol-resistant strains.

  • 1.

    Angelo KM, Reynolds J, Karp BE, Hoekstra RM, Scheel CM, Friedman C, 2016. Antimicrobial resistance among nontyphoidal Salmonella isolated from blood in the United States, 2003–2013. J Infect Dis 214: 15651570.

    • Search Google Scholar
    • Export Citation
  • 2.

    Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA, 2012. Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 379: 24892499.

    • Search Google Scholar
    • Export Citation
  • 3.

    Dong GX, Wen SL, 2018. Research advancement on the economic impact of antimicrobial resistance. Chin Pharm J 5: 330334.

  • 4.

    Aminov RI, Mackie RI, 2007. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett 271: 147161.

  • 5.

    Leversteinvan Hall MA 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 17: 873880.

    • Search Google Scholar
    • Export Citation
  • 6.

    Lazarus B, Paterson DL, Mollinger JL, Rogers BA, 2015. Do human extraintestinal Escherichia coli infections resistant to expanded-spectrum cephalosporins originate from food-producing animals? A systematic review. Clin Infect Dis 60: 439452.

    • Search Google Scholar
    • Export Citation
  • 7.

    Bonten MJ, Mevius D, 2015. Less evidence for an important role of food-producing animals as source of antibiotic resistance in humans. Clin Infect Dis 60: 1867.

    • Search Google Scholar
    • Export Citation
  • 8.

    Schwarz S, Kehrenberg C, Doublet B, Cloeckaert A, 2004. Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol Rev 28: 519542.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lu J 2018. Spread of the florfenicol resistance floR gene among clinical Klebsiella pneumoniae isolates in China. Antimicrob Resist Infect Control 7: 127.

    • Search Google Scholar
    • Export Citation
  • 10.

    Liu WW, Wang Y, 2018. Mechanisms of bacterial resistance to florfenicol. Chin J Anim Infect Dis 1: 16.

  • 11.

    Zhan Z, Kuang D, Liao M, Zhang H, Lu J, Hu X, Ye Y, Meng J, Xu X, Zhang J, 2017. Antimicrobial susceptibility and molecular typing of Salmonella Senftenberg isolated from humans and other sources in Shanghai, China, 2005 to 2011. J Food Protection 80: 146.

    • Search Google Scholar
    • Export Citation
  • 12.

    Zhao Q, Wang Y, Wang S, Wang Z, Du XD, Jiang H, Xia X, Shen Z, Ding S, Wu C, 2016. Prevalence and abundance of florfenicol and linezolid resistance genes in soils adjacent to swine feedlots. Sci Rep 6: 32192.

    • Search Google Scholar
    • Export Citation
  • 13.

    Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ, 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 3: 5967.

    • Search Google Scholar
    • Export Citation
  • 14.

    Li C, Chen J, Wang J, Ma Z, Han P, Luan Y, Lu A, 2015. Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment. Sci Total Environ 522: 101107.

    • Search Google Scholar
    • Export Citation
  • 15.

    Silbergeld EK, Graham J, Price LB, 2008. Industrial food animal production, antimicrobial resistance, and human health. Annu Rev Public Health 29: 151169.

    • Search Google Scholar
    • Export Citation
  • 16.

    Kim JS, Yun YS, Kim SJ, Jeon SE, Lee DY, Chung GT, Yoo CK, Kim J, Group PNKW, 2016. Rapid emergence and clonal dissemination of CTX-M-15-producing Salmonella enterica serotype Virchow, South Korea. Emerg Infect Dis 22: 68.

    • Search Google Scholar
    • Export Citation
  • 17.

    Zhang QQ, Ying GG, Pan CG, Liu YS, Zhao JL, 2015. Comprehensive evaluation of antibiotics emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. Environ Sci Technol 49: 67726782.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rapid Emergence of Florfenicol-Resistant Invasive Non-Typhoidal Salmonella in China: A Potential Threat to Public Health

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  • 1 National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Key Laboratory of Zoonoses, Ministry of Agriculture, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Animal Infectious Diseases Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China;
  • 2 Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China;
  • 3 Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, China

Infection caused by invasive Salmonella occurs when Salmonella bacteria, which normally cause diarrhea, enter the bloodstream and spread through the body. We report the dramatic increase in florfenicol-resistant invasive non-typhoidal Salmonella (iNTS) in China between 2007 and 2016. Of the 186 iNTS strains isolated during the study period, 34 were florfenicol resistant, most of which harbored known resistance genes. Florfenicol is exclusively used in veterinary medicine in China, but now florfenicol-resistant iNTS is found in clinical patients. This finding indicates that antimicrobial resistance produced in veterinary medicine can be transmitted to humans, which poses a severe threat to public health.

INTRODUCTION

Invasive non-typhoidal Salmonella (iNTS) infections pose a serious threat to public health. Particularly, in sub-Saharan Africa, iNTS has become a common cause of bacterial infections resulting in death.1 According to previous reports, the extensive use of antimicrobials has led to a rapid increase in antimicrobial resistance in iNTS, which is of great concern.2 It is estimated that by 2050, 10 million people will die each year because of bacterial resistance, and the cumulative cost of global resistance will reach U.S.$100 trillion.3 So, it is critical to understand the origin and transmission pathways of antimicrobial resistance to help predict the occurrence and spread of clinically relevant pathogens.4

Numerous researchers have hypothesized that antimicrobial-resistant bacteria can be transmitted from veterinary medicine to humans.5 However, whether antimicrobial-resistant strains generated in animals represent an origin for human infections has been controversial for many years.6,7 Florfenicol (FFC) is an antimicrobial that is exclusively used in veterinary medicine in China and many other countries.8,9 The extensive use of FFC has resulted in the emergence and increase in florfenicol-resistant strains of bacteria found in animals, which causes great loss to the breeding industry.10 However, the potential risks of FFC use in animals to public health remain unknown, and it is critical to research this. This study investigated florfenicol resistance in iNTS isolated from clinical patients in China, to survey the potential threat to public health from the use of FFC.

MATERIALS AND METHODS

Ethics statement.

Ethical approval for this study was provided by Shanghai Municipal Center for Disease Control and Prevention (Shanghai, China).

Sample collection and antimicrobial susceptibility testing.

One hundred eighty-six iNTS strains were collected from clinical patient samples, including blood, aspirates, and cerebrospinal fluid, in Shanghai, Xinjiang, Inner Mongolia, Henan, Guangxi, Zhejiang, Chongqing, and Fujian Provinces in China between 2007 and 2016. Serovars were assigned according to the Kauffmann–White scheme. Antimicrobial susceptibility tests were performed on the 186 iNTS strains using an agar dilution method to test resistance against 14 antimicrobial agents according to the guidelines of Clinical and Laboratory Standards Institute standards.11 The tested antimicrobial agents included the following: ampicillin (AMP), cefotaxime (CTX) (third-generation cephalosporin), cefepime (CFP) (fourth-generation cephalosporin), streptomycin (STR), sulfisoxazole (SUL), nalidixic acid (NA), ofloxacin (OFX), ciprofloxacin (CIP), chloramphenicol (C), imipenem (IPM), polymyxin B (PB), tetracycline (TET), enrofloxacin (ENR), and FFC. Escherichia coli ATCC25922 and ATCC35218 were used as quality control strains. Interpretations of results were made according to the Clinical Laboratory Standards Institute criteria.11

Molecular analysis of florfenicol resistance.

Salmonella strains exhibiting florfenicol resistance were analyzed by polymerase chain reaction (PCR) assays. The known florfenicol resistance genes fexA, fexB, pexA, floR, cfr, and optrA were screened by PCR using previously described primers.12

Pulsed-field gel electrophoresis (PFGE).

Pulsed-field gel electrophoresis was conducted with restriction enzyme XbaI to establish genetic relationships of the 34 florfenicol-resistant iNTS strains according to a standardized protocol.13

Statistical analysis.

Comparison of frequencies was calculated using the chi-squared test using SAS 9.2 (SAS Institute, Cary, NC). A P-value < 0.05 was considered to indicate statistical significance.

RESULTS

Sample collection and antimicrobial susceptibility testing.

Results showed the highest resistance rates were to NA (61.8%), followed by SUL (58.1%), STR (44.6%), TET (41.4%), C (29.6%), FFC (18.3%), AMP (9.7%), CTX (8.1%), ENR (3.8%), CIP (2.7%), OFX (2.2%), CFP (1.6%), and PB (1.6%). All iNTS strains exhibited susceptibility to IPM. Multidrug resistance, defined as resistance to ≥ 3 antimicrobial agents, was found in 101 strains (54.3%) (Table 1).

Table 1

Antimicrobial susceptibility of 186 invasive Salmonella strains from humans in China, 2007–2016

No. of strainsNo. of strains resistant to indicated agent at the indicated breakpoint in mg/L (% resistance)
Multidrug resistance phenotypeCiprofloxacinCefotaximeCefepimePolymyxin BOfloxacinEnrofloxacinAmpicillinSulfisoxazoleChloramphenicolTetracyclineNalidixic acidFlorfenicolStreptomycin
≥ 4≥ 4≥ 32≥ 8≥ 8≥ 4≥ 32≥ 512≥ 32≥ 16≥ 32≥ 16≥ 64
186101 (54.3)5 (2.7)15 (8.1)3 (1.6)3 (1.6)4 (2.2)7 (3.8)18 (9.7)108 (58.1)55 (29.6)77 (41.4)115 (61.8)34 (18.3)83 (44.6)

Of the 34 florfenicol-resistant strains, 32 (94.1%) were multidrug resistant and two (5.9%) were resistant to two antimicrobial agents. The first florfenicol-resistant iNTS strain in our study was isolated in 2010. Since then, 2, 1, 3, 5, 8, and 14 strains were isolated in 2011, 2012, 2013, 2014, 2015, and 2016, respectively (Figure 1). The rates of florfenicol-resistant iNTS have increased significantly, from 0% in 2007–2009 to 9.1% (1/11) in 2010 and 2011, 8.3% (1/12) in 2012, 10.7% (3/28) in 2013, 22.7% (5/22) in 2014, 32% (8/25) in 2015, and 30.4% (14/46) in 2016. The percentage of florfenicol-resistant rates in the year 2016 (14/46, P < 0.05) was significantly higher than that of 2007 (0/14).

Figure 1.
Figure 1.

Trend of florfenicol resistance of invasive non-typhoidal Salmonella strains from humans in China (2007–2016). Denominator in brackets indicate the total number of blood culture–positive non-typhoidal Salmonella strains cultured in each year. The numerator in brackets indicates the florfenicol-resistant strains.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0403

Molecular analysis of florfenicol resistance.

floR was the most commonly detected gene, found in 25 strains. fexB and fexA were observed in three and two strains, respectively (Figure 2).

Figure 2.
Figure 2.

XbaI pulsed-field gel electrophoresis profiles and detection of resistance genes of 34 florfenicol-resistant strains.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0403

Pulsed-field gel electrophoresis.

There were 34 PFGE patterns generated among the 34 iNTS strains. The strains were divided into seven clusters (A–G) and 16 individual distinct profiles at 80% profile similarity. Some strains of the same serotypes recovered from the same or different regions at various times in clusters C, D, F, and G had high genetic similarity (≥ 80%) (Figure 2). Cluster C was the biggest cluster and comprised five strains, including four strains recovered from Shanghai and one strain isolated from Guangxi. Cluster E involved strains from Shanghai, Guangxi, and Chongqing. Cluster F included strains from Shanghai and Chongqing.

DISCUSSION

Our results support the theory that antimicrobial resistance in veterinary medicine presents a serious threat to human health. Florfenicol is exclusively used in veterinary medicine in China,9 but now florfenicol-resistant iNTS is found in clinical patients. China is the largest producer and consumer of antimicrobials in the world, and 210,000 tons of antimicrobials are produced annually, of which 48% are used for veterinary medicine. Around 80% of the antimicrobials sold in the United States are used in animals, which makes breeding farms an important source of antimicrobial-resistant bacterial pathogens.14,15 More importantly, most antimicrobials such as quinolone and cephalosporin drugs are simultaneously used for human and veterinary medicine, which means resistant strains of animal origin are also resistant to human antimicrobials, increasing the difficulty of antimicrobial treatment in human bacterial infections.16

Three genes under study in our review (floR, fexB, and fexA) have been reported in bacteria of animal origin and encode florfenicol resistance by activating the active efflux pump mechanism to expel the drug from the extracellular domain.12,17 These genes have been identified in plasmids in florfenicol-resistant bacteria, which means that using florfenicol in veterinary medicine can result in selection of important, easily transmitted, resistant phenotypes.10,12

Clusters C, D, F, and G had high genetic similarity (≥ 80%) (Figure 2), which shows the potential for horizontal transmission of resistance and cross-infection. The identification of 16 distinct PFGE profiles of florfenicol-resistant strains indicates that they were diverse, and florfenicol-resistant strains were found to harbor the related resistance genes that the transmission of florfenical-resistant iNTS from animals to humans has been widespread.

The current study highlights that of 186 strains isolated between 2007 and 2016, a total of 34 were resistant to FFC. Since the first isolation of florfenicol-resistant strains in 2010, their prevalence has dramatically increased, from 0% in the year 2007, 9.1% in 2010, and up to 30.4% in 2016. Our findings indicate that although FFC is exclusively used in veterinary medicine in China, florfenicol-resistant iNTS strains can be transmitted to humans. This supports that antimicrobial resistance produced in veterinary medicine can be transmitted to human pathogens, which is an urgent threat to human health. Comprehensive surveillance of antimicrobial use and resistance in veterinary medicine is required, and the specific mode of transmission of florfenicol resistance between animals and human pathogens needs further study.

REFERENCES

  • 1.

    Angelo KM, Reynolds J, Karp BE, Hoekstra RM, Scheel CM, Friedman C, 2016. Antimicrobial resistance among nontyphoidal Salmonella isolated from blood in the United States, 2003–2013. J Infect Dis 214: 15651570.

    • Search Google Scholar
    • Export Citation
  • 2.

    Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA, 2012. Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 379: 24892499.

    • Search Google Scholar
    • Export Citation
  • 3.

    Dong GX, Wen SL, 2018. Research advancement on the economic impact of antimicrobial resistance. Chin Pharm J 5: 330334.

  • 4.

    Aminov RI, Mackie RI, 2007. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett 271: 147161.

  • 5.

    Leversteinvan Hall MA 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 17: 873880.

    • Search Google Scholar
    • Export Citation
  • 6.

    Lazarus B, Paterson DL, Mollinger JL, Rogers BA, 2015. Do human extraintestinal Escherichia coli infections resistant to expanded-spectrum cephalosporins originate from food-producing animals? A systematic review. Clin Infect Dis 60: 439452.

    • Search Google Scholar
    • Export Citation
  • 7.

    Bonten MJ, Mevius D, 2015. Less evidence for an important role of food-producing animals as source of antibiotic resistance in humans. Clin Infect Dis 60: 1867.

    • Search Google Scholar
    • Export Citation
  • 8.

    Schwarz S, Kehrenberg C, Doublet B, Cloeckaert A, 2004. Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol Rev 28: 519542.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lu J 2018. Spread of the florfenicol resistance floR gene among clinical Klebsiella pneumoniae isolates in China. Antimicrob Resist Infect Control 7: 127.

    • Search Google Scholar
    • Export Citation
  • 10.

    Liu WW, Wang Y, 2018. Mechanisms of bacterial resistance to florfenicol. Chin J Anim Infect Dis 1: 16.

  • 11.

    Zhan Z, Kuang D, Liao M, Zhang H, Lu J, Hu X, Ye Y, Meng J, Xu X, Zhang J, 2017. Antimicrobial susceptibility and molecular typing of Salmonella Senftenberg isolated from humans and other sources in Shanghai, China, 2005 to 2011. J Food Protection 80: 146.

    • Search Google Scholar
    • Export Citation
  • 12.

    Zhao Q, Wang Y, Wang S, Wang Z, Du XD, Jiang H, Xia X, Shen Z, Ding S, Wu C, 2016. Prevalence and abundance of florfenicol and linezolid resistance genes in soils adjacent to swine feedlots. Sci Rep 6: 32192.

    • Search Google Scholar
    • Export Citation
  • 13.

    Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ, 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 3: 5967.

    • Search Google Scholar
    • Export Citation
  • 14.

    Li C, Chen J, Wang J, Ma Z, Han P, Luan Y, Lu A, 2015. Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment. Sci Total Environ 522: 101107.

    • Search Google Scholar
    • Export Citation
  • 15.

    Silbergeld EK, Graham J, Price LB, 2008. Industrial food animal production, antimicrobial resistance, and human health. Annu Rev Public Health 29: 151169.

    • Search Google Scholar
    • Export Citation
  • 16.

    Kim JS, Yun YS, Kim SJ, Jeon SE, Lee DY, Chung GT, Yoo CK, Kim J, Group PNKW, 2016. Rapid emergence and clonal dissemination of CTX-M-15-producing Salmonella enterica serotype Virchow, South Korea. Emerg Infect Dis 22: 68.

    • Search Google Scholar
    • Export Citation
  • 17.

    Zhang QQ, Ying GG, Pan CG, Liu YS, Zhao JL, 2015. Comprehensive evaluation of antibiotics emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. Environ Sci Technol 49: 67726782.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Ming Liao or Jianmin Zhang, College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Rd., Tianhe District, Guangzhou 510642, China. E-mails: mliao@scau.edu.cn or junfeng-v@163.com

Financial support: This work was supported by the National Key R&D Program of China (2017YFC1600101, 2018YFD0500500); a project supported by the National Natural Science Foundation of China (31972762); a project supported by Guangdong Province Universities and Colleges Pearl River Scholar–Funded Scheme (2018); Pearl River S&T Nova Program of Guangzhou (201806010183); Province Science and Technology of Guangdong Research Project (2017A020208055); and Guangdong Key S&T Program (Grant no. 2019B020217002).

Authors’ addresses: Zeqiang Zhan, Yuan Gao, Fanliang Zeng, Xiaoyun Qu, Hongxia Zhang, Ming Liao, and Jianmin Zhang, National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Key Laboratory of Zoonoses, Ministry of Agriculture, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Animal Infectious Diseases Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China, E-mails: zeqiangzhan_v@163.com, tianc__001@163.com, 1529436709@qq.com, junjingzhang1989@163.com, qxy0926@126.com, mliao@scau.edu.cn, and junfeng-v@163.com. Xuebin Xu, Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China, E-mail: xxb72@sina.com. Haiyan Shen, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, China, E-mail: haiyan_0001@163.com.

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