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Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses
International Journal of Food Microbiology 84 (2003) 27 – 32 www.elsevier.com/locate/ijfoodmicro
Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses A. Martel *, L.A. Devriese, A. Decostere, F. Haesebrouck Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 18 February 2002; received in revised form 5 July 2002; accepted 18 August 2002
Abstract Macrolide and lincosamide (ML)-resistant streptococci and enterococci from tonsillar and colon swabs from 33 pigs and 99 pork carcasses swabs from animals originating from different farms in Belgium were isolated, and their ML resistance phenotypes and genotypes were determined by disk diffusion test and PCR assay, amplifying the ermB gene and the mefA gene. From each of the 33 pigs and 88 of the 99 carcasses’ swabs, at least one resistant strain was isolated. The predominant phenotype was the constitutively expressed macrolides, lincosamides and streptogramin B (MLSB) phenotype. This phenotype was most often encoded by the ermB gene. A minority of the strains showed the M phenotype encoded by the mefA gene in streptococci, or the L or ML phenotype. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Streptococci; Enterococci; Pig; Pork carcasses; Macrolide resistance; ErmB
1. Introduction Resistance to all major groups of antibiotics has arisen hand in hand with their extensive use in medicine and animal husbandry, and macrolide and lincosamide antibiotics are no exception (Aarestrup et al., 1998a,b; Jensen et al., 1999; Portillo et al., 2000; Butaye et al., 2001). Commercially available macrolides can be divided into 14 (erythromycin, clarithromycin)-, 15 (azithromycin)-, and 16 (tylosine, spiramycin)-membered lactone ring macrolides. Lincosamide antibiotics are lincomycin, clindamycin and pirlimycin. The strep* Corresponding author. Tel.: +32-9-2647435; fax: +32-92647494. E-mail address: [email protected] (A. Martel).
togramin antibiotics are composed of two factors, A and B (M and S in virginiamycine, respectively) that act in synergy. Use of the streptogramin antibiotics as growth-promoter antibiotics is no longer permitted in the EU, but the macrolides are increasingly applied especially in pigs (FIDIN Antibioticarapportage, 2001). Macrolide, lincosamide and streptogramin antibiotics are chemically distinct but have similar mode of action. Therefore, they are commonly indicated as the macrolide – lincosamide – streptogramin (MLS) family. A large array of resistance genes has been described (Roberts et al., 1999) and many of these confer cross-resistance to several or most antibiotics of this family. Resistance to macrolides is based on three different mechanisms: (1) target modification by point mutation or methylation of 23S rRNA, (2) inactivation of the
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(02)00390-2
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A. Martel et al. / International Journal of Food Microbiology 84 (2003) 27–32
antibiotic and (3) presence of efflux pumps removing the antibiotic internally from the bacteria (Leclercq and Courvalin, 1991; Weisblum, 1995). According to the published literature, the most frequently found macrolide resistance genes in bacterial isolates from animals and humans are the erm (erythromycin ribosome methylation) genes (Roberts et al., 1999). These genes encode methyltransferases that target specific residues in 23S rRNA. Presence of an Erm methylase confers resistance to macrolides, lincosamides and streptogramin B (MLSB phenotype). The phenotypic expression of target site modification can be inducible or constitutive. Different classes of erm genes have been described, but in streptococci and enterococci, the ermB gene is most frequently encountered (Arpin et al., 1999; Jensen et al., 1999; Roberts et al., 1999). Drug efflux by a membrane-bound protein encoded by a mef (macrolide efflux) gene is also an important mechanism of resistance against macrolides in streptococci. This mechanism results in the M resistance phenotype and involves resistance to 14- and 15membered macrolide compounds. However, the strains remain susceptible to 16-membered macrolide, lincosamide and streptogramin B antibiotics (Roberts et al., 1999). It was the aim of the present study to determine the presence of macrolide and lincosamide resistance in streptococci and enterococci isolated from pigs and pork carcasses. Both resistance phenotypes and genotypes were determined.
2. Materials and methods 2.1. Samples Tonsillar and colon swabs were collected from 33 pigs, originating from 33 different farms in Belgium, and were brought in for necropsy. Ninety-nine swabs were taken by swabbing the entire skin surface of pork carcasses immediately after slaughter in four slaughterhouses. The pigs originated from 35 different Belgian farms. The swabs were inoculated on Columbia agar (Oxoid, Basingstoke, England) supplemented with 5% sheep blood, colistin and aztreonam, and Slanetz and Bartley (Oxoid) agar, to isolate streptococci and enter-
ococci. Three plates of both agars were supplemented with 1 Ag/ml erythromycin, 8 Ag/ml erythromycin or 10 Ag/ml lincomycin, respectively. Plates were incubated at 37 jC in a 5% CO2-enriched environment for 24 and 48 h. All different types of colonies were purified. 2.2. Identification Representative isolates were identified using tDNA-PCR (Baele et al., 2000, 2001). To discriminate between Streptococcus dysgalactiae subsp. dysgalactiae and equisimilis, Lancefield typing and streptokinase activity was determined (Vandamme et al., 1996). From each animal or carcass, only one strain per species carrying the same resistance phenotype and genotype was included in the results. 2.3. Phenotype determination Antimicrobial susceptibility patterns of streptococci were determined by disk diffusion on Columbia blood agar, while enterococci were tested on ISO agar (Oxoid). The antimicrobial test tablets clindamycin, erythromycin, lincomycin and tylosin, all obtained from Rosco Diagnostics, Taastrup, Denmark, were used. Bacterial suspensions were prepared in 0.9% NaCl to a 0.5 McFarland standard from growth on Columbia blood agar after overnight incubation at 37 jC in a 5% CO2-enriched environment. The inhibition zone diameters were measured after overnight incubation at 37 jC in 5% CO2 atmosphere and interpreted following the manufacturer’s instructions. MLS resistance phenotypes were recorded as described by Leclercq and Courvalin (1991). An MLSB phenotype isolate was defined as an isolate that was resistant to erythromycin, clindamycin, lincomycin and tylosin. An M phenotype isolate was an isolate found to be resistant to erythromycin alone, and the L phenotype showed resistance to lincosamides only. 2.4. PCR The primers for ermB (5VGAAAAGA/GTACTCAACCAAATA3Vand 5VAGTAACGGTACTTAAATTGTTTAC3V) and mefA (5VAGTATCATTAATCACTAGTGC3V and 5VTTCTTCTGGTACTAAAAGTGG3V) were derived from published sequences (Sutcliffe et al., 1996a,b) to provide specific PCR products of 640
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29
and 400 bp, respectively. PCR conditions for the detection of the ermB and mefA genes were described previously (Martel et al., 2001).
Table 2 Resistance phenotype and genotype from enterococci isolated from tonsillar and colon swabs from 33 pigs from 33 different farms
3. Results
Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus
3.1. Strains 3.1.1. Resistant streptococci and enterococci from tonsillar and colon swabs of pigs The findings obtained with resistant streptococci and enterococci isolated from tonsillar and colon swabs of 33 pigs are summarized in Tables 1 and 2. From each of the 33 pigs, at least one resistant streptococcal species was isolated. Streptococcus alactolyticus, Streptococcus gallolyticus and Streptococcus bovis were only isolated from the colon swabs. Only one S. dysgalactiae and one Streptococcus pluranimalium strain were isolated from tonsillar swabs. Streptococcus hyointestinalis and Streptococcus suis strains were recovered from tonsillar as well as from colon swabs. Macrolide- and lincosamide-resistant enterococci were isolated from 24 out of the 33 pigs examined. Enterococcus faecalis and Enterococcus hirae were the most frequently found species. Only two strains (one Enterococcus faecium and one E. hirae) were obtained from the tonsil samples.
Table 1 Resistance phenotype and genotype from streptococci isolated from tonsillar and colon swabs from 33 pigs from 33 different farms Isolated Number Number Number Number from MLSB ermB + mefA + Streptococcus alactolyticus Streptococcus bovis Streptococcus dysgalactiae Streptococcus gallolyticus Streptococcus hyointestinalis Streptococcus pluranimalium Streptococcus suis
C
9
9
9
0
C
4
2
2
0
T
1
1
1
0
C
8
8
8
0
C, T
4
4
4
0
T
1
1
1
0
33
33
32
1
C, T
C—colon swab, T—tonsillar swab.
avium cecorum durans faecalis faecium hirae
Number
Number MLSB
Number ermB +
Number mefA +
6 1 1 14 8 10
4 1 1 14 8 10
3 1 1 14 7 8
0 0 0 0 0 0
3.1.2. Resistant streptococci and enterococci isolated from pork carcasses Neither resistant enterococci nor streptococci were isolated from 11 out of 99 pork carcasses swabs. S. suis and S. alactolyticus were the most prevalent species (Table 3). Thirteen out of 23 S. dysgalactiae strains belonged to Lancefield group C, none to Lancefield group G. None of the strains showed streptokinase activity, which allows them to be identified as S. dysgalactiae subsp. dysgalactiae. Resistant enterococci were isolated from only 13 pork carcasses (Table 4). E. faecium was more often found than others. 3.2. Phenotype determination 3.2.1. Resistant streptococci and enterococci from tonsillar and colon swabs of pigs Fifty-eight out of 60 isolated streptococci (97%) and 38 out of 40 enterococci (95%) were resistant to erythromycin, tylosin, lincomycin and clindamycin, which corresponds to the MLSB phenotype (Tables 1 Table 3 Resistance phenotype and genotype from streptococci isolated from 99 pork carcasses, originating from 35 different farms at four slaughterhouses Number Number Number Number MLSB ermB + mefA + Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus
agalactiae alactolyticus bovis dysgalactiae hyointestinalis pneumoniae suis hyovaginalis porcinus
1 42 7 23 11 4 43 2 3
1 34 7 15 11 4 41 1 2
1 33 7 13 8 3 40 1 2
0 1 0 8 2 1 3 0 0
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Table 4 Resistance phenotype and genotype from enterococci isolated from 99 pork carcasses, originating from 35 different farms at four slaughterhouses Number Number Number Number MLSB ermB + mefA + Enterococcus Enterococcus Enterococcus Enterococcus
faecalis faecium hirae gallinarum
4 8 1 2
3 7 1 1
3 6 1 0
0 0 0 0
and 2). No inducible resistance was seen. Two S. bovis strains showed the L phenotype. Two Enterococcus avium strains were susceptible to erythromycin and resistant to tylosin, clindamycin and lincomycin. 3.2.2. Resistant streptococci and enterococci isolated from pork carcasses One hundred and sixteen out of 136 streptococci (85%) and 12 out of 15 enterococci were resistant to all the tested antimicrobials (MLSB phenotype) (Tables 3 and 4). The MLSB phenotype was constitutively expressed. Seven streptococcal isolates (two S. suis and five S. dysgalactiae strains) showed the M phenotype. Twelve strains (eight S. alactolyticus, two S. dysgalactiae, one Streptococcus hyovaginalis and one Streptococcus porcinus isolate) were resistant to lincomycin and clindamycin, but remained susceptible to the macrolides tested (L phenotype). One S. dysgalactiae strain was resistant to erythromycin, lincomycin and clindamycin, but susceptible to tylosin. Three enterococci (one E. faecalis, one E. faecium and one Enterococcus gallinarum) were resistant to the lincosamides and susceptible to the macrolides (L phenotype). 3.3. Resistance genotype 3.3.1. Resistant streptococci and enterococci from tonsillar and colon swabs of pigs Fifty-seven out of the 58 MLSB-resistant streptococci carried the ermB gene (Table 1). The mefA gene was detected in one S. suis strain with the MLSB phenotype. Thirty-four out of the 38 MLSB-resistant enterococci possessed the ermB gene (Table 2). One E. avium, one E. faecium and two E. hirae strains with
the MLSB phenotype were negative in the PCR reaction. 3.3.2. Resistant streptococci and enterococci isolated from pork carcasses One hundred and eight out of the 116 streptococci with the MLSB phenotype were positive for the ermB gene. One Streptococcus pneumoniae, one S. dysgalactiae, one S. suis and two S. hyointestinalis with the MLSB phenotype carried the mefA gene, but not ermB. Both genes were detected in one S. alactolyticus and one S. dysgalactiae strain, each with the MLSB phenotype. Three strains (one S. alactolyticus, one S. dysgalactiae and one S. hyointestinalis) with the MLSB phenotype gave negative PCR results for both genes. Five S. dysgalactiae and two S. suis strains with the M phenotype were positive for the mefA gene. The mefA gene was also found in one S. dysgalactiae strain resistant to erythromycin, lincomycin and clindamycin. Ten out of 12 MLSB-resistant enterococci possessed the ermB gene. One E. faecium and one E. gallinarum with the MLSB phenotype were negative in the PCR reaction.
4. Discussion In this study, macrolide- and lincosamide-resistant streptococci and enterococci were frequently found in tonsillar and colon swabs of pigs and on pork carcasses. Pork carcasses can be contaminated by tonsillar or faecal pig flora, but also slaughterhouse workers or other sources of ‘in-house’ bacteria can contaminate the carcasses. This was confirmed in this study by isolating S. pneumoniae strains from carcass swabs. Interestingly, S. dysgalactiae was isolated much more often from pork carcasses than from tonsil and colon samples. S. dysgalactiae consists of two subspecies of which subsp. equisimilis is associated with humans, and subsp. dysgalactiae with animals (Vandamme et al., 1996). The former contains mainly Lancefield group G and C strains, while porcine strains belonging to the latter are h-hemolytic group C and L strains. Animal group G strains belong to the species Streptococcus canis (Devriese et al., 1986). In order to exclude possible human origin of the isolated
A. Martel et al. / International Journal of Food Microbiology 84 (2003) 27–32
S. dysgalactiae strains, the Lancefield type and streptokinase activity of these strains were determined (Vandamme et al., 1996), since tDNA-PCR (Baele et al., 2000, 2001) does not allow discrimination between S. dysgalactiae subsp. dysgalactiae and equisimilis. None of the isolated S. dysgalactiae strains belonged to Lancefield group G and none of these strains showed streptokinase activity. This allows them to be identified as S. dysgalactiae subsp. dysgalactiae. Therefore, we conclude that they were from animal origin. The predominant resistance phenotype was the MLSB phenotype (co-resistance between macrolides, lincosamides and streptogramin B antibiotics). We performed the disk diffusion test to detect the inducible macrolide resistance phenotype (Leclercq and Courvalin, 1991), but no such strains were found. These results are in agreement with our previous results in S. suis isolates recovered from diseased pigs (Martel et al., 2001). A minority of the strains showed the M, L and ML phenotype. A similar distribution of phenotype patterns was obtained by Lagrou et al. (2000) with Belgian S. pneumoniae isolates. In human strains, there is a geographical difference in the prevalence of the M phenotype, which is common in the USA and Canada, but appears to be rare in Belgium (Sutcliffe et al., 1996a,b; Johnston et al., 1998; Lagrou et al., 2000). No data of resistance phenotypes in strains from pigs from the USA or Canada are available. The MLSB phenotype was found to be encoded mainly by the ermB gene, which is in agreement with the results of Jensen et al. (1999) obtained in a study of enterococcal and streptococcal strains from pigs, cattle, broiler chickens and humans. In human streptococci in Belgium, ermB-encoded resistance is also the most important mechanism in S. pneumoniae and Streptococcus pyogenes (Descheemaeker et al., 2000; Lagrou et al., 2000). In a limited number of the streptococcal strains, the presence of the mefA gene was demonstrated. Relatively, more mefA positive strains were found on pork carcasses than in tonsillar and colon swabs. This might be due to the higher incidence of S. dysgalactiae in the carcasses since the mefA gene was demonstrated relatively often (8/24) in these strains. In some strains, the resistance phenotype was not in complete agreement with the resistance genotype.
31
For example, a S. suis strain with the MLSB phenotype was only positive for the mefA gene, which indicates that the strain has at least one other resistance mechanism that is responsible for the lincosamide resistance. It is believed that resistant strains pass from animal to man and that resistance can spread to man by direct contact or by eating meat products. Since the ermB gene is frequently found in porcine and in human strains, it might be possible that this gene is transferred between animal and human strains. In a previous study, we detected an identical sequence for a 540-bp fragment of the ermB gene in a limited number of porcine (S. suis) and human (S. pneumoniae and S. pyogenes) streptococcal strains (Martel et al., 2001). Further studies, including sequencing the entire ermB gene, are required to obtain better insights into possible exchange of resistance genes between human and porcine streptococcal strains. The presence of identical mobile DNA elements in human and porcine strains would also indicate horizontal transfer of these resistance genes. Acknowledgements This work was supported by the Ministry of Agriculture (DG6), Brussels, Belgium. The technical assistance of Els Verleysen is gratefully appreciated. Tonsillar and colon swabs from pigs were kindly provided by J. Hommez, Regional Veterinary Investigation Centre, Torhout. References Aarestrup, F.M., Jorsal, S.E., Jensen, N.E., 1998a. Serological characterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996. Vet. Microbiol. 60, 59 – 66. Aarestrup, F.M., Bager, F., Jensen, N.E., Madsen, M., Meyling, A., Wegener, H.C., 1998b. Surveillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark. APMIS 16, 606 – 622. Arpin, C., Daube, H., Tessier, F., Quentin, C., 1999. Presence of mefA and mefE genes in Streptococcus agalactiae. Antimicrob. Agents Chemother. 43, 944 – 946. Baele, M., Baele, P., Vaneechoutte, M., Storms, V., Butaye, P., Devriese, L.A., Verschraegen, G., Gillis, M., Haesebrouck, F., 2000. Application of tRNA intergenic spacer PCR for identi-
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fication of Enterococcus species. J. Clin. Microbiol. 38, 4201 – 4207. Baele, M., Storms, V., Haesebrouck, F., Devriese, L.A., Gillis, M., Verschraegen, G., De Baere, T., Vaneechoutte, M., 2001. Application and evaluation of the interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) for identification of Streptococcus species. J. Clin. Microbiol. 39, 1436 – 1442. Butaye, P., Devriese, L.A., Haesebrouck, F., 2001. Differences in antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium strains isolated from farm and pet animals. Antimicrob. Agents Chemother. 45, 1374 – 1378. Descheemaeker, P., Chapelle, S., Lammens, C., Hauchecorne, M., Wijdooghe, M., Vandamme, P., Ieven, M., Goossens, H., 2000. Macrolide resistance and erythromycine resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates. J. Antimicrob. Chemother. 45, 167 – 173. Devriese, L.A., Hommez, J., Kilpper-Ba¨lz, R., Schleifer, K.H., 1986. Streptococcus canis sp. nov.: a species of group G streptococci from animals. Int. J. Syst. Bacteriol. 36, 422 – 425. FIDIN Antibioticarapportage, 2001. Tijdschr. Diergeneeskd. 126, 560 – 562. Jensen, L.B., Frimodt-Moller, N., Aarestrup, F.M., 1999. Presence of erm gene classes in Gram-positive bacteria of animal and human origin in Denmark. FEMS Microbiol. Lett. 170, 151 – 158. Johnston, N.J., De, A.J., Kellner, J.D., Low, D.E., 1998. Prevalence and characterization of the mechanisms of macrolide, lincosamide, and streptogramin resistance in isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42, 2425 – 2426. Lagrou, K., Peetermans, W.E., Verhaegen, J., Van Lierde, S., Verbist, L., Van Eldere, J., 2000. Macrolide resistance in Belgian
Streptococcus pneumoniae. J. Antimicrob. Chemother. 45, 119 – 121. Leclercq, L., Courvalin, P., 1991. Bacterial resistance to macrolide, lincosamide and streptogramin antibiotics by target modification. Antimicrob. Agents Chemother. 35, 1267 – 1272. Martel, A., Baele, M., Devriese, L.A., Goossens, H., Wisselink, H.J., Decostere, A., Haesebrouck, F., 2001. Prevalence and mechanism of resistance against macrolides and lincosamides in Streptococcus suis isolates. Vet. Microbiol. 83, 287 – 297. Portillo, A., Ruiz-Larrea, F., Zaragaza, M., Alonso, A., Martinez, J.L., Torres, C., 2000. Macrolide resistance genes in Enterococcus spp. Antimicrob. Agents Chemother. 44, 967 – 971. Roberts, M.C., Sutcliffe, J., Courvalin, P., Jensen, L.B., Rood, J., Seppala, H., 1999. Minireview. Nomenclature for macrolide and macrolide – lincosamide – streptogramin B resistance determinants. Antimicrob. Agents Chemother. 43, 2823 – 2830. Sutcliffe, J., Grebe, T., Tait-Kamradt, A., Wondrack, L., 1996a. Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agents Chemother. 40, 2562 – 2566. Sutcliffe, J., Tait-Kamradt, A., Wondrack, L., 1996b. Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycine: a common resistance pattern mediated by an efflux system. Antimicrob. Agents Chemother. 40, 1817 – 1824. Vandamme, P., Pot, B., Falsen, E., Kersters, K., Devriese, L.A., 1996. Taxonomic study of Lancefield streptococcal groups C, G, and L (Streptococcus dysgalactiae) and proposal of S. dysgalactiae subsp. Equisimilis subsp. nov. Int. J. Syst. Bacteriol. 46, 774 – 781. Weisblum, B., 1995. Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother. 39, 577 – 585.
Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses A. Martel *, L.A. Devriese, A. Decostere, F. Haesebrouck Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 18 February 2002; received in revised form 5 July 2002; accepted 18 August 2002
Abstract Macrolide and lincosamide (ML)-resistant streptococci and enterococci from tonsillar and colon swabs from 33 pigs and 99 pork carcasses swabs from animals originating from different farms in Belgium were isolated, and their ML resistance phenotypes and genotypes were determined by disk diffusion test and PCR assay, amplifying the ermB gene and the mefA gene. From each of the 33 pigs and 88 of the 99 carcasses’ swabs, at least one resistant strain was isolated. The predominant phenotype was the constitutively expressed macrolides, lincosamides and streptogramin B (MLSB) phenotype. This phenotype was most often encoded by the ermB gene. A minority of the strains showed the M phenotype encoded by the mefA gene in streptococci, or the L or ML phenotype. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Streptococci; Enterococci; Pig; Pork carcasses; Macrolide resistance; ErmB
1. Introduction Resistance to all major groups of antibiotics has arisen hand in hand with their extensive use in medicine and animal husbandry, and macrolide and lincosamide antibiotics are no exception (Aarestrup et al., 1998a,b; Jensen et al., 1999; Portillo et al., 2000; Butaye et al., 2001). Commercially available macrolides can be divided into 14 (erythromycin, clarithromycin)-, 15 (azithromycin)-, and 16 (tylosine, spiramycin)-membered lactone ring macrolides. Lincosamide antibiotics are lincomycin, clindamycin and pirlimycin. The strep* Corresponding author. Tel.: +32-9-2647435; fax: +32-92647494. E-mail address: [email protected] (A. Martel).
togramin antibiotics are composed of two factors, A and B (M and S in virginiamycine, respectively) that act in synergy. Use of the streptogramin antibiotics as growth-promoter antibiotics is no longer permitted in the EU, but the macrolides are increasingly applied especially in pigs (FIDIN Antibioticarapportage, 2001). Macrolide, lincosamide and streptogramin antibiotics are chemically distinct but have similar mode of action. Therefore, they are commonly indicated as the macrolide – lincosamide – streptogramin (MLS) family. A large array of resistance genes has been described (Roberts et al., 1999) and many of these confer cross-resistance to several or most antibiotics of this family. Resistance to macrolides is based on three different mechanisms: (1) target modification by point mutation or methylation of 23S rRNA, (2) inactivation of the
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(02)00390-2
28
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antibiotic and (3) presence of efflux pumps removing the antibiotic internally from the bacteria (Leclercq and Courvalin, 1991; Weisblum, 1995). According to the published literature, the most frequently found macrolide resistance genes in bacterial isolates from animals and humans are the erm (erythromycin ribosome methylation) genes (Roberts et al., 1999). These genes encode methyltransferases that target specific residues in 23S rRNA. Presence of an Erm methylase confers resistance to macrolides, lincosamides and streptogramin B (MLSB phenotype). The phenotypic expression of target site modification can be inducible or constitutive. Different classes of erm genes have been described, but in streptococci and enterococci, the ermB gene is most frequently encountered (Arpin et al., 1999; Jensen et al., 1999; Roberts et al., 1999). Drug efflux by a membrane-bound protein encoded by a mef (macrolide efflux) gene is also an important mechanism of resistance against macrolides in streptococci. This mechanism results in the M resistance phenotype and involves resistance to 14- and 15membered macrolide compounds. However, the strains remain susceptible to 16-membered macrolide, lincosamide and streptogramin B antibiotics (Roberts et al., 1999). It was the aim of the present study to determine the presence of macrolide and lincosamide resistance in streptococci and enterococci isolated from pigs and pork carcasses. Both resistance phenotypes and genotypes were determined.
2. Materials and methods 2.1. Samples Tonsillar and colon swabs were collected from 33 pigs, originating from 33 different farms in Belgium, and were brought in for necropsy. Ninety-nine swabs were taken by swabbing the entire skin surface of pork carcasses immediately after slaughter in four slaughterhouses. The pigs originated from 35 different Belgian farms. The swabs were inoculated on Columbia agar (Oxoid, Basingstoke, England) supplemented with 5% sheep blood, colistin and aztreonam, and Slanetz and Bartley (Oxoid) agar, to isolate streptococci and enter-
ococci. Three plates of both agars were supplemented with 1 Ag/ml erythromycin, 8 Ag/ml erythromycin or 10 Ag/ml lincomycin, respectively. Plates were incubated at 37 jC in a 5% CO2-enriched environment for 24 and 48 h. All different types of colonies were purified. 2.2. Identification Representative isolates were identified using tDNA-PCR (Baele et al., 2000, 2001). To discriminate between Streptococcus dysgalactiae subsp. dysgalactiae and equisimilis, Lancefield typing and streptokinase activity was determined (Vandamme et al., 1996). From each animal or carcass, only one strain per species carrying the same resistance phenotype and genotype was included in the results. 2.3. Phenotype determination Antimicrobial susceptibility patterns of streptococci were determined by disk diffusion on Columbia blood agar, while enterococci were tested on ISO agar (Oxoid). The antimicrobial test tablets clindamycin, erythromycin, lincomycin and tylosin, all obtained from Rosco Diagnostics, Taastrup, Denmark, were used. Bacterial suspensions were prepared in 0.9% NaCl to a 0.5 McFarland standard from growth on Columbia blood agar after overnight incubation at 37 jC in a 5% CO2-enriched environment. The inhibition zone diameters were measured after overnight incubation at 37 jC in 5% CO2 atmosphere and interpreted following the manufacturer’s instructions. MLS resistance phenotypes were recorded as described by Leclercq and Courvalin (1991). An MLSB phenotype isolate was defined as an isolate that was resistant to erythromycin, clindamycin, lincomycin and tylosin. An M phenotype isolate was an isolate found to be resistant to erythromycin alone, and the L phenotype showed resistance to lincosamides only. 2.4. PCR The primers for ermB (5VGAAAAGA/GTACTCAACCAAATA3Vand 5VAGTAACGGTACTTAAATTGTTTAC3V) and mefA (5VAGTATCATTAATCACTAGTGC3V and 5VTTCTTCTGGTACTAAAAGTGG3V) were derived from published sequences (Sutcliffe et al., 1996a,b) to provide specific PCR products of 640
A. Martel et al. / International Journal of Food Microbiology 84 (2003) 27–32
29
and 400 bp, respectively. PCR conditions for the detection of the ermB and mefA genes were described previously (Martel et al., 2001).
Table 2 Resistance phenotype and genotype from enterococci isolated from tonsillar and colon swabs from 33 pigs from 33 different farms
3. Results
Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus
3.1. Strains 3.1.1. Resistant streptococci and enterococci from tonsillar and colon swabs of pigs The findings obtained with resistant streptococci and enterococci isolated from tonsillar and colon swabs of 33 pigs are summarized in Tables 1 and 2. From each of the 33 pigs, at least one resistant streptococcal species was isolated. Streptococcus alactolyticus, Streptococcus gallolyticus and Streptococcus bovis were only isolated from the colon swabs. Only one S. dysgalactiae and one Streptococcus pluranimalium strain were isolated from tonsillar swabs. Streptococcus hyointestinalis and Streptococcus suis strains were recovered from tonsillar as well as from colon swabs. Macrolide- and lincosamide-resistant enterococci were isolated from 24 out of the 33 pigs examined. Enterococcus faecalis and Enterococcus hirae were the most frequently found species. Only two strains (one Enterococcus faecium and one E. hirae) were obtained from the tonsil samples.
Table 1 Resistance phenotype and genotype from streptococci isolated from tonsillar and colon swabs from 33 pigs from 33 different farms Isolated Number Number Number Number from MLSB ermB + mefA + Streptococcus alactolyticus Streptococcus bovis Streptococcus dysgalactiae Streptococcus gallolyticus Streptococcus hyointestinalis Streptococcus pluranimalium Streptococcus suis
C
9
9
9
0
C
4
2
2
0
T
1
1
1
0
C
8
8
8
0
C, T
4
4
4
0
T
1
1
1
0
33
33
32
1
C, T
C—colon swab, T—tonsillar swab.
avium cecorum durans faecalis faecium hirae
Number
Number MLSB
Number ermB +
Number mefA +
6 1 1 14 8 10
4 1 1 14 8 10
3 1 1 14 7 8
0 0 0 0 0 0
3.1.2. Resistant streptococci and enterococci isolated from pork carcasses Neither resistant enterococci nor streptococci were isolated from 11 out of 99 pork carcasses swabs. S. suis and S. alactolyticus were the most prevalent species (Table 3). Thirteen out of 23 S. dysgalactiae strains belonged to Lancefield group C, none to Lancefield group G. None of the strains showed streptokinase activity, which allows them to be identified as S. dysgalactiae subsp. dysgalactiae. Resistant enterococci were isolated from only 13 pork carcasses (Table 4). E. faecium was more often found than others. 3.2. Phenotype determination 3.2.1. Resistant streptococci and enterococci from tonsillar and colon swabs of pigs Fifty-eight out of 60 isolated streptococci (97%) and 38 out of 40 enterococci (95%) were resistant to erythromycin, tylosin, lincomycin and clindamycin, which corresponds to the MLSB phenotype (Tables 1 Table 3 Resistance phenotype and genotype from streptococci isolated from 99 pork carcasses, originating from 35 different farms at four slaughterhouses Number Number Number Number MLSB ermB + mefA + Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus
agalactiae alactolyticus bovis dysgalactiae hyointestinalis pneumoniae suis hyovaginalis porcinus
1 42 7 23 11 4 43 2 3
1 34 7 15 11 4 41 1 2
1 33 7 13 8 3 40 1 2
0 1 0 8 2 1 3 0 0
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Table 4 Resistance phenotype and genotype from enterococci isolated from 99 pork carcasses, originating from 35 different farms at four slaughterhouses Number Number Number Number MLSB ermB + mefA + Enterococcus Enterococcus Enterococcus Enterococcus
faecalis faecium hirae gallinarum
4 8 1 2
3 7 1 1
3 6 1 0
0 0 0 0
and 2). No inducible resistance was seen. Two S. bovis strains showed the L phenotype. Two Enterococcus avium strains were susceptible to erythromycin and resistant to tylosin, clindamycin and lincomycin. 3.2.2. Resistant streptococci and enterococci isolated from pork carcasses One hundred and sixteen out of 136 streptococci (85%) and 12 out of 15 enterococci were resistant to all the tested antimicrobials (MLSB phenotype) (Tables 3 and 4). The MLSB phenotype was constitutively expressed. Seven streptococcal isolates (two S. suis and five S. dysgalactiae strains) showed the M phenotype. Twelve strains (eight S. alactolyticus, two S. dysgalactiae, one Streptococcus hyovaginalis and one Streptococcus porcinus isolate) were resistant to lincomycin and clindamycin, but remained susceptible to the macrolides tested (L phenotype). One S. dysgalactiae strain was resistant to erythromycin, lincomycin and clindamycin, but susceptible to tylosin. Three enterococci (one E. faecalis, one E. faecium and one Enterococcus gallinarum) were resistant to the lincosamides and susceptible to the macrolides (L phenotype). 3.3. Resistance genotype 3.3.1. Resistant streptococci and enterococci from tonsillar and colon swabs of pigs Fifty-seven out of the 58 MLSB-resistant streptococci carried the ermB gene (Table 1). The mefA gene was detected in one S. suis strain with the MLSB phenotype. Thirty-four out of the 38 MLSB-resistant enterococci possessed the ermB gene (Table 2). One E. avium, one E. faecium and two E. hirae strains with
the MLSB phenotype were negative in the PCR reaction. 3.3.2. Resistant streptococci and enterococci isolated from pork carcasses One hundred and eight out of the 116 streptococci with the MLSB phenotype were positive for the ermB gene. One Streptococcus pneumoniae, one S. dysgalactiae, one S. suis and two S. hyointestinalis with the MLSB phenotype carried the mefA gene, but not ermB. Both genes were detected in one S. alactolyticus and one S. dysgalactiae strain, each with the MLSB phenotype. Three strains (one S. alactolyticus, one S. dysgalactiae and one S. hyointestinalis) with the MLSB phenotype gave negative PCR results for both genes. Five S. dysgalactiae and two S. suis strains with the M phenotype were positive for the mefA gene. The mefA gene was also found in one S. dysgalactiae strain resistant to erythromycin, lincomycin and clindamycin. Ten out of 12 MLSB-resistant enterococci possessed the ermB gene. One E. faecium and one E. gallinarum with the MLSB phenotype were negative in the PCR reaction.
4. Discussion In this study, macrolide- and lincosamide-resistant streptococci and enterococci were frequently found in tonsillar and colon swabs of pigs and on pork carcasses. Pork carcasses can be contaminated by tonsillar or faecal pig flora, but also slaughterhouse workers or other sources of ‘in-house’ bacteria can contaminate the carcasses. This was confirmed in this study by isolating S. pneumoniae strains from carcass swabs. Interestingly, S. dysgalactiae was isolated much more often from pork carcasses than from tonsil and colon samples. S. dysgalactiae consists of two subspecies of which subsp. equisimilis is associated with humans, and subsp. dysgalactiae with animals (Vandamme et al., 1996). The former contains mainly Lancefield group G and C strains, while porcine strains belonging to the latter are h-hemolytic group C and L strains. Animal group G strains belong to the species Streptococcus canis (Devriese et al., 1986). In order to exclude possible human origin of the isolated
A. Martel et al. / International Journal of Food Microbiology 84 (2003) 27–32
S. dysgalactiae strains, the Lancefield type and streptokinase activity of these strains were determined (Vandamme et al., 1996), since tDNA-PCR (Baele et al., 2000, 2001) does not allow discrimination between S. dysgalactiae subsp. dysgalactiae and equisimilis. None of the isolated S. dysgalactiae strains belonged to Lancefield group G and none of these strains showed streptokinase activity. This allows them to be identified as S. dysgalactiae subsp. dysgalactiae. Therefore, we conclude that they were from animal origin. The predominant resistance phenotype was the MLSB phenotype (co-resistance between macrolides, lincosamides and streptogramin B antibiotics). We performed the disk diffusion test to detect the inducible macrolide resistance phenotype (Leclercq and Courvalin, 1991), but no such strains were found. These results are in agreement with our previous results in S. suis isolates recovered from diseased pigs (Martel et al., 2001). A minority of the strains showed the M, L and ML phenotype. A similar distribution of phenotype patterns was obtained by Lagrou et al. (2000) with Belgian S. pneumoniae isolates. In human strains, there is a geographical difference in the prevalence of the M phenotype, which is common in the USA and Canada, but appears to be rare in Belgium (Sutcliffe et al., 1996a,b; Johnston et al., 1998; Lagrou et al., 2000). No data of resistance phenotypes in strains from pigs from the USA or Canada are available. The MLSB phenotype was found to be encoded mainly by the ermB gene, which is in agreement with the results of Jensen et al. (1999) obtained in a study of enterococcal and streptococcal strains from pigs, cattle, broiler chickens and humans. In human streptococci in Belgium, ermB-encoded resistance is also the most important mechanism in S. pneumoniae and Streptococcus pyogenes (Descheemaeker et al., 2000; Lagrou et al., 2000). In a limited number of the streptococcal strains, the presence of the mefA gene was demonstrated. Relatively, more mefA positive strains were found on pork carcasses than in tonsillar and colon swabs. This might be due to the higher incidence of S. dysgalactiae in the carcasses since the mefA gene was demonstrated relatively often (8/24) in these strains. In some strains, the resistance phenotype was not in complete agreement with the resistance genotype.
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For example, a S. suis strain with the MLSB phenotype was only positive for the mefA gene, which indicates that the strain has at least one other resistance mechanism that is responsible for the lincosamide resistance. It is believed that resistant strains pass from animal to man and that resistance can spread to man by direct contact or by eating meat products. Since the ermB gene is frequently found in porcine and in human strains, it might be possible that this gene is transferred between animal and human strains. In a previous study, we detected an identical sequence for a 540-bp fragment of the ermB gene in a limited number of porcine (S. suis) and human (S. pneumoniae and S. pyogenes) streptococcal strains (Martel et al., 2001). Further studies, including sequencing the entire ermB gene, are required to obtain better insights into possible exchange of resistance genes between human and porcine streptococcal strains. The presence of identical mobile DNA elements in human and porcine strains would also indicate horizontal transfer of these resistance genes. Acknowledgements This work was supported by the Ministry of Agriculture (DG6), Brussels, Belgium. The technical assistance of Els Verleysen is gratefully appreciated. Tonsillar and colon swabs from pigs were kindly provided by J. Hommez, Regional Veterinary Investigation Centre, Torhout. References Aarestrup, F.M., Jorsal, S.E., Jensen, N.E., 1998a. Serological characterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996. Vet. Microbiol. 60, 59 – 66. Aarestrup, F.M., Bager, F., Jensen, N.E., Madsen, M., Meyling, A., Wegener, H.C., 1998b. Surveillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark. APMIS 16, 606 – 622. Arpin, C., Daube, H., Tessier, F., Quentin, C., 1999. Presence of mefA and mefE genes in Streptococcus agalactiae. Antimicrob. Agents Chemother. 43, 944 – 946. Baele, M., Baele, P., Vaneechoutte, M., Storms, V., Butaye, P., Devriese, L.A., Verschraegen, G., Gillis, M., Haesebrouck, F., 2000. Application of tRNA intergenic spacer PCR for identi-
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