|Brief communication||Peer reviewed|
To investigate occurrence of acquired resistance, minimum inhibitory concentrations of florfenicol for Streptococcus suis isolated in France between 2011 and 2014 were determined. No acquired resistance to florefenicol was observed among recent field isolates of S suis after more than 10 years of use of this antibiotic.
Para investigar la ocurrencia de la resistencia adquirida, las concentraciones mínimas inhibitorias de florfenicol para aislamientos de Streptoccocus suis en Francia entre 2011 y 2014 fueron determinados. No se observó resistencia adquirida al florfenicol entre aislamientos de campo recientes de S suis después de más de 10 años de uso de este antibiótico.
Afin d’étudier l’occurrence de résistance acquise envers le florfénicol, on détermina les concentrations minimales inhibitrices d’isolats de Streptococcus suis obtenus entre 2011 et 2014 d’élevages en France. Aucune résistance acquise au florfénicol ne fut observée parmi les isolats récents de S suis après plus de 10 ans d’utilisation de cet antibiotique
Keywords: swine, swine respiratory disease, Streptococcus suis, florfenicol
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Received: July 7, 2014
Accepted: October 28, 2014
Streptococcus suis is a major pathogen in swine production, causing meningitis, arthritis, septicemia, bronchopneumonia, polyserositis, and endocarditis.1 It is also recognized as an important zoonotic agent.2
A florfenicol concentrate solution is labelled in the United States for treatment of swine respiratory disease (SRD) associated with several bacterial pathogens, including S suis. Treatment is administered by the oral route through drinking water. In France, florfenicol is approved for treatment and control of respiratory disease caused by the major pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida, but S suis is not included in the claim. However, S suis may occur simultaneously or sequentially with such bacteria in SRD and is also found in the upper respiratory tracts of healthy animals.3 Thus S suis may be exposed to florfenicol during treatment of animals suffering from SRD caused by bacterial pathogens.
The objective of this study was to determine susceptibility to florfenicol of recent S suis field isolates from pig herds in western France to determine whether acquired resistance has emerged. Minimum inhibitory concentrations (MICs) were determined to provide epidemiological survey data.
Materials and methods
Bacteria were isolated at the Institut en Santé Agro-Environnement laboratory (Public Veterinary Diagnostic Laboratory, Fougeres, France) between 2011 and 2014 from samples submitted for disease diagnosis in piglets from herds located in the west of France (mainly Ille-et-Vilaine, but also in Brittany, Pays de la Loire, and Normandy). Isolation, identification, and serotyping of isolates were conducted by conventional bacteriological methods: culture, Gram staining, biochemical tests (Api 20 Strep strip; BioMérieux, Marcy l’Etoile, France) and serotyping (S suis antisera; LDA 22, Ploufragan, France).
All isolates were stored at < -60°C in brain-heart broth with 15% glycerol until MIC determination. Determination of MICs was performed on independent S suis isolates from an epidemiological point of view (ie, no more than one isolate per year per herd).
Minimal inhibitory concentrations were determined using a broth microdilution method. Testing was performed according to Clinical and Laboratory Standards Institute guidelines (CLSI VET01-A44 and VET01-S25). Briefly, after isolates were incubated overnight on agar plates and purity of the cultures was confirmed, the direct colony suspension method was used. Bacterial suspensions were adjusted in cation-adjusted Mueller-Hinton broth supplemented with 2.5% lysed horse blood. The final concentration in the 50 µL of bacterial suspension added per microtiter plate well was approximately 5 × 105 colony forming units (CFU) per mL. Minimum inhibitory concentrations were determined using 96-well microtiter plates containing dehydrated antibiotic (CMP1ASPV, florfenicol custom veterinary susceptibility plate format; Trek Diagnostic Systems Inc, Cleveland, Ohio). After incubation for 24 hours at 35°C (standard deviation 2°C) in ambient air, the MIC was recorded as the lowest concentration of florfenicol that completely inhibited growth of the organism in wells, as detected by the unaided eye.
As directed in CLSI guidelines,4,5 Enterococcus faecalis ATCC 29212 and Streptococcus pneumoniae ATCC 49619 were used as reference strains for MIC quality controls each day of testing, with acceptable quality control ranges 2 to 8 µg per mL and 1 to 4 µg per mL, respectively. A colony count of the inoculum for each plate was performed to ensure that the final inoculum in wells approximated 5 × 105 CFU per mL.
The 45 S suis isolates used in the study were from routine submissions to the Public Veterinary Diagnostic Laboratory, from piglets 4 to 9 weeks old suffering from respiratory disease (19 isolates; 42%), endocarditis (9 isolates; 20%), septicemia (8 isolates; 18%), meningitis (5 isolates; 11%), and arthritis (4 isolates; 9%). Isolates belonged to different serotypes: type 2 (38% of isolates), non-typeable (24%), 7 (13%), 1,2 (7%), 3 (7%), 1 (4%), 8 (4%), and 9 (2%). Minimum inhibitory concentrations of florfenicol ranged from 0.5 to 2 µg per mL; MIC50 and MIC90 were 1 and 2 µg per mL, respectively, confirming the previous data, as shown in Table 1.
Table 1: Florfenicol MIC50, MIC90, and MIC ranges reported for Streptococcus suis isolates*
|Susceptibility of S suis isolates to florfenicol†||Concentrations of florfenicol (µg/mL)‡|
|S suis isolation||MIC50 (µg/mL)||MIC90 (µg/mL)||MIC ranges (µg/mL)|
|Callens et al6||2010||ND||ND||0.5-8||0.03-128|
|Portis et al7||2007-2010||2||2||0.06->32||0.06-32|
|Schwarz and Kehrenberg8||2000-2005||1||2||0.25-2||0.125-128|
|Kehrenberg et al9||2002-2003||1||2||0.25-2||0.125-128|
|Priebe and Schwarz10||2000-2001||1||2||0.25-2||0.125-128|
|Wisselink et al11||1987-1997||ND||ND||0.5-4||0.06-32|
* Areas of isolation: West of France (present study); Belgium (Callens); North America: United States and Canada (Portis); Germany (Schwarz,
Kehrenberg, Priebe); Belgium, UK, France, Italy, Spain, Germany, and The Netherlands (Wisselink).
† MIC determination methods: Broth microdilution for the present study and all references except Callens6 (agar dilution).
‡ Range of concentrations of florfenicol tested.
MIC = Minimum inhibitory concentration; ND = not determined
All isolates included in this study were considered to be susceptible to florfenicol, according to CLSI VET01-S2-approved breakpoints for S suis, which are ≤ 2 µg per mL (susceptible), 4 µg per mL (intermediate), and ≥ 8 µg per mL (resistant).5 Data provided by this study are consistent with results of previous studies in which resistance was not detected among isolates collected in France until 200212 and in Germany between 2000 and 2005.8-10
Streptococcus suis isolates classified as intermediate to florfenicol have seldom been isolated in Europe11 or North America.7 According to the studies of Callens et al6 and Portis et al,7 resistance to florfenicol is seldom if ever reported (one isolate among 331 European and approximatively 2000 American isolates tested), whereas the susceptibilities of S suis isolates to several antibiotics (erythromycin, lincomycin, penicillin, tiamulin, tetracycline, tilmicosin, and tylosin) have dramatically decreased in Europe over the past few years.6
Distribution of isolates among serotypes is in accordance with previous French data, but with a higher percentage of non-typeable isolates and a lower percentage of serotype 9.13 Wisselink et al11 could not show an association between the serotype of an isolate and its susceptibility pattern. The number of isolates in this study was too small to confirm this result.
Although no resistance to florfenicol was found among the S suis isolates tested in this study, the authors do not recommend extra-label use of florfenicol.
Under the conditions of this study, S suis isolates collected from piglets in the west of France between 2011 and 2014 were not resistant to florfenicol.
Conflict of interest
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 information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.
1. Staats JJ, Feder I, Okwumabua O, Chengappa MM. Streptococcus suis: past and present. Vet Res Commun.1997;21:381–407.
2. Lun ZR, Wang QP, Chen XG, Li AX, Zhu XQ. Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis. 2007;7:201–209.
3. Amass SF, Wu CF, Clark LK. Evaluation of antibiotics for the elimination of the tonsillar carrier state of Streptococcus suis in pigs. J Vet Diagn Invest. 1996;8:64–67.
4. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard – Fourth Edition. CLSI document VET01-A4. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 2013.
5. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Second Information Supplement. CLSI document VET01-S2. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 2013.
6. Callens BF, Haesebrouck F, Maes D, Butaye P, Dewulf J, Boyen F. Clinical resistance and decreased susceptibility in Streptococcus suis isolates from clinically healthy fattening pigs. Microb Drug Resist. 2013;2:146–151.
7. Portis E, Lindeman C, Johansen L, Stoltman G. Antimicrobial susceptibility of porcine Pasteurella multocida, Streptococcus suis, and Actinobacillus pleuropneumoniae from the United States and Canada, 2001 to 2010. J Swine Health Prod. 2013;21:30–41.
*8. Schwarz S, Kehrenberg C. Florfenicol susceptibility of porcine respiratory tract pathogens in Germany 2000-2005. Proc 19th IPVS Cong. Copenhagen, Denmark. 2006;2:437. Abstract P31-04.
9. Kehrenberg C, Mumme J, Wallmann J, Verspohl J, Tegeler R, Kühn T, Schwarz S. Monitoring of florfenicol susceptibility among bovine and porcine respiratory tract pathogens collected in Germany during the years 2002 and 2003. J Antimicrob Chemother. 2004;54:572–574.
10. Priebe S, Schwarz S. In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob Agents Chemother. 2003;47:2703–2705.
11. Wisselink HJ, Veldman KT, Van den Eede C, Salmon SA, Mevius DJ. Quantitative susceptibility of Streptococcus suis strains isolated from diseased pigs in seven European countries to antimicrobial agents licenced in veterinary medicine. Vet Microbiol. 2006;113:73–82.
12. Marie J, Morvan H, Berthelot-Herault F, Sanders P, Kempf I, Gautier-Bouchardon AV, Jouy E, Kobisch M. Antimicrobial susceptibility of Streptococcus suis isolated from swine in France and from humans in different countries between 1996 and 2000. J Antimicrob Chemother. 2002;50:201–209.
*13. Gottschalk M, Kobisch M, Berthelot-Herault F. L’infection à Streptococcus suis chez le porc [Streptococcus suis infection in swine]. Proc Journées de la Recherche Porcine en France. Paris, France. 2001;33:269–276.
* Non-refereed references.