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Mutations in the quinolone-resistance determining region (QRDR) of Salmonella strains isolated from pigs in Spain
Veterinary Microbiology 106 (2005) 297–301 www.elsevier.com/locate/vetmic
Short communication
Mutations in the quinolone-resistance determining region (QRDR) of Salmonella strains isolated from pigs in Spain C. Seminati a,b,*, W. Mejia a, E. Mateu a,b, M. Martin a,b a
Departament de Sanitat i d’Anatomia Animals, Universitat Auto`noma de Barcelona (UAB), Facultat de Veterinaria, 08193 Bellaterra (Cerdanyola del Valle`s), Barcelona, Spain b Centre de Recerca en Sanitat Animal (CReSA), Universitat Auto`noma de Barcelona (UAB), 08193 Bellaterra (Cerdanyola del Valle`s), Barcelona, Spain Received 23 June 2004; received in revised form 9 December 2004; accepted 23 December 2004
Abstract Quinolone-resistance determining region (QRDR) of Gyrase A gene was sequenced in 54 Salmonella strains of pig origin that have different quinolone-resistance patterns. Those strains accounted for 12 different serotypes. Mutations at Ser83 or Asp87 were predominant in the studied isolates. However, for serotypes Anatum and Virchow, resistance to quinolones seemed to be linked to specific mutations, namely, Ser83 ! Tyr and Ser83 ! Phe, respectively. Other mutations found in different positions did not seem to have clinical significance except for changes at Asp82. # 2005 Elsevier B.V. All rights reserved. Keywords: Quinolone; Resistance; QRDR; Salmonella; Pig
1. Introduction Nowadays, an intense debate on the causes of the increasing antimicrobial resistance is being held among scientists. It has been suggested that the non-therapeutic use of antimicrobial agents in animals is one of the main factors that contribute to the emergence of drug resistances. Particularly, there is a concern for the development of resistant zoonotic bacteria such as Salmonella that may pass to people * Corresponding author. Tel.: +34 93 5811046; fax: +34 93 5813297. E-mail address: [email protected] (C. Seminati).
through the food chain and can produce untreatable disease. For example, a Salmonella strain of pig origin with decreased susceptibility to quinolones caused a severe outbreak of salmonellosis in Denmark where two people died (Mølbak et al., 1999). Fluoroquinolones have become the first choice treatment for septicemic salmonellosis and thus, the emergence of quinolone-resistant Salmonella strains causes a considerable concern. Acquired resistance to fluoroquinolones arises from several mechanisms: mutations of the target enzymes, modification of membrane porins or overproduction of proteins involved in efflux systems such as the AcrAB-TolC (Everett et al., 1996; Hooper,
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.12.024
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2001a, 2001b; Baucheron et al., 2004). In Gram negative bacteria, quinolone resistance is usually conferred by point mutations in target enzymes, especially those encoding for the DNA gyrase (gyrA, gyrB, parC, parE). For Salmonella, mutations in the gyrA gene, that encodes for the subunit A of the DNA gyrase, seem to be responsible for the development of most resistances to quinolones. These mutations have been clustered in a region of the gene product between amino acids 67 and 122, the so-called quinoloneresistance determining region (QRDR) (Deguchi et al., 1996; Ferrero et al., 1994). Single amino acid changes at Ser-83 (change by Phe, Tyr, or Ala) or at Asp-87 (change by Gly, Asn, or Tyr) are the most frequent (Griggs et al., 1996). However, some authors suggested that this QRDR can be larger and could start at position 51 (Friedman et al., 2001). In the present study, we investigated the mutations in the QRDR of Salmonella isolates of porcine origin in order to figure out their distribution and significance.
Virchow (5), Anatum (4), Brandenburg (4), Hadar (3), Enteritidis (3), and one strain each of serotypes Abony, Bredeney, Grumpensis and Choleraesuis. According to the available data, isolates were epidemiologically unrelated. Selected isolates were cultured overnight in BHI broth at 37 8C. Then, cultures were centrifuged and suspended in 1 ml of Rnase and Dnase free water (Ambion). DNA extraction was done by boiling for 10 min. Amplification and sequencing of the gyrA QRDR region was done according to a previously described PCR protocol (Walker et al., 2001) modified from Griggs et al. (1996). With this technique the amplicon contained a region between codons 37 and 151 of the gyrA gene. PCR products were then sequenced using an ABI 373 A Stretch sequencer. Predicted amino acid sequences were compared to a quinolone-susceptible reference strain LT2 (GenBank accession number AE008801)
3. Results 2. Materials and methods Fifty-four Salmonella strains were selected from a bacterial collection of pig isolates of Catalonia (Spain) obtained from 1998 to 2002. Selected strains were chosen representing the most frequent pig isolates in Spain for that period. Susceptibility profiles of these isolates have been described before (Mateu et al., 2002; Mejia et al., 2003). Briefly, strains were classified for quinolone resistance according to the results of Kirby–Bauer disk diffusion test (nalidixic acid, and ciprofloxacin) or by the minimal inhibitory concentration determined in a microdilution test (enrofloxacin). All susceptibility tests were done according to NCCLS recommendations (NCCLS, 2002). Fourteen strains were susceptible to all quinolones. The remaining 40 strains were resistant or had decreased susceptibility to 1 or more drugs. For nalidixic acid, 37 strains were resistant (inhibition diameter 13 mm) and 3 were classified as intermediate (14–18 mm). For enrofloxacin 3 strains were resistant (MIC > 2.0 mg/ml) and 31 were intermediate (MIC > 0.5 and 2.0 mg/ml); for ciprofloxacin only 2 strains were intermediate (16–20 mm) and all others were susceptible. Distribution of isolates by serotype was: Typhimurium (15), Tilburg (9), 4,5,12:i:- (7),
Mutations in the gyrA gene were found in 42 strains (77.7%). The most frequent mutation (n = 23) corresponded to codon at position 87 (Asp ! Asn, 21 strains; Asp ! Tyr, two strains), followed by changes at position 83 (n = 9) (Ser ! Phe, six strains; Ser ! Tyr, three strains). Changes Asp87 ! Tyr were found only in serotype Enteriditis (two isolates) while all Ser83 ! Tyr changes were found only in serotype Anatum (three isolates). Five of the six strains with a change Ser83 ! Phe belonged to serotype Virchow. In one case, the recently described mutation at position 82 (Asp ! Arg) was found in one isolate of serotype Tilburg (MIC for enrofloxacin = 1.3 mg/ ml) that also had a mutation in Asp87 ! Asn. None of the strains had a double Ser83/Asp87 mutation. We also found other mutations in the gyrA QRDR that were not described before. These mutations could be found alone or in association with other changes. Thus, 16 strains mutated in position 78 (His ! Asn or less commonly His ! Asp). Four strains had a Pro79 ! His mutation, three a Gly107 ! Cys change and two strains had mutations in position 64 (Lys ! Asn and Lys ! Ser). These changes were found in several serotypes regardless whether or not a given strain was susceptible or resistant to quinolones.
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299
Table 1 Serovars and susceptibility profiles against quinolones for Salmonella isolates of pig origin with mutations in QRDR codons Ser83 or Asp87 Serovar
Disk diffusion test Nal
Cip
Virchow1 Virchow Virchow Virchow Virchow Tilburg Anatum Anatum Anatum 4.5.12:i:Hadar Hadar Tilburg Tilburg2 Tilburg Tilburg3 Tilburg Tilburg4 Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Tilburg Enteritidis Enteritidis
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S R R
S S S S S S S S S S S S I S S S S S I S S S S S S S S S S S S S
MIC (mg/ml), Enr 0.7 0.7 0.7 0.7 1.3 0.7 1.3 NT 1.3 1.3 0.7 0.7 2.3 1.3 1.3 2.3 2.3 1.3 1.3 1.3 0.7 1.3 1.3 0.7 1.3 1.3 0.7 0.3 1.3 0.3 NT 0.7
Mutations in QRDR of gyrA Ser83
Asp87
Phe Phe Phe Phe Phe Phe Tyr Tyr Tyr Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Tyr Tyr
His78
Pro79
Asn Asn
His
Asp
Asp
Asn
Asn
Asp Asn
His His
QRDR: quinolone-resistance determining region; MIC: minimal inhibitory concentration. Nal: nalidixic acid; Cip: ciprofloxacin; Enr: enrofloxacin; S: susceptible; R: resistant; I: intermediate. Isolates that had other mutations—superscript 1: His80 ! Arg, Gln106 ! Leu; superscript 2: Asp82 ! Arg; superscript 3: Lys64 ! Asn; superscript 4 Gly107 ! Cys.
Twelve isolates had no mutations in the gyrA gene, but 2 were intermediate to enrofloxacin (MIC = 1.3 mg/ ml) and 2 more were susceptible to enrofloxacin (MIC = 0.1 mg/ml) although resistant to nalidixic acid. These results are summarized in Tables 1 and 2.
4. Discussion In our study we focused on gyrA mutations of Salmonella strains of pig origin that had been classified as resistant to nalidixic acid. Thirty-two of the examined strains had mutations in the clinically
significant positions of gyrA Ser83 and Asp87 but none had a double Ser83/Asp87 mutation. Nevertheless, some of them were resistant to enrofloxacin (MIC > 2.0 mg/ml). This fact indicates either that this single gyrA mutation can confer a high level of resistance, or that additional mechanisms were involved, for example, mutations in gyrB, parC or parE genes. However, the involvement of parC, parE and gyrB mutations in the development of quinolone resistance for salmonellae is controversial and some authors (Ling et al., 2003) suggested that parC and parE mutations appear mainly after gyrA changes developed, played thus a secondary role. Recently,
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Table 2 Serovars and susceptibility profiles against quinolones for Salmonella isolates of pig origin with mutations in QRDR other than Ser83 or Asp87 or without mutations Serovar
Disk diffusion test 1
Bredeney 4.5.12:i:Brandenburg Brandenburg Brandenburg Anatum 4.5.12:i:Enteriditis Tilburg Typhimurium2 4.5.12:i:Brandenburg Typhimurium Typhimurium 4.5.12:i:4.5.12:i:Choleraesuis Typhimurium Hadar Abony Grumpensis 4.5.12:i:-
Nal
Cip
S R I I I S S S R S R R R R S S S S S S S S
S S S S S S S S S S S S S S S S S S S S S S
MIC (mg/ml), Enr 0.3 0.1 1.3 1.3 1.3 0.3 0.1 0.1 1.3 0.1 0.1 1.3 0.1 1.3 0.1 0.1 0.3 0.1 NT 0.1 0.3 0.1
Mutations in QRDR of gyrA His78
Gly107
Asn Asn Asn Asn Asn Asn Asn Asn
Cys
Pro79
Lys64 Ser
His Cys
QRDR: quinolone-resistance determining region; MIC: minimal inhibitory concentration. Nal: nalidixic acid; Cip: ciprofloxacin; Enr: enrofloxacin; S: susceptible; R: resistant; I: intermediate. Isolates that had other mutations: superscript 1: Gln106 ! His; superscript 2: Gly108 ! Cys.
Eaves et al. (2004) described that Salmonella enterica strains with double gyrA and parC mutations were more susceptible to ciprofloxacin than other similar strains having a single gyrA mutation suggesting some kind of compensatory mechanism. It is interesting to note that some amino acid substitutions were only found in certain serotypes. That was the case for Ser83 ! Tyr in Salmonella Anatum and Ser ! Phe in Salmonella Virchow. In previous studies (Griggs et al., 1996; Piddock et al., 1998), the change Ser83 ! Phe has been reported for animal isolates of Salmonella Enteritidis. However, other changes have been not associated with a given serotype. The isolates that we studied were epidemiologically unrelated and, for that reason, it would be tempting to suggest that, in pigs, a given gyrA mutation can be associated to a given serotype. Notwithstanding, a larger study would be required to confirm this observation.
We also observed point mutations not described before. The most frequent ones corresponded to positions Lys64, His78, Pro79 and Gly107. These mutations were found regardless of the quinolonesusceptibility profile and we think that they are not related to the development of resistance. Regarding the strains that did not show any mutation, some were resistant to either nalidixic acid or were intermediate to enrofloxacin, a result that further indicates the existence of additional resistance mechanisms. However, the involvement of gyrB or parC mutations could not be ruled out (Allen and Poppe, 2002). Our results showed that Ser83 and Asp87 mutations seem to be one of the main mechanisms for the development of quinolone resistance in Salmonella of pig origin, but other mechanisms could be involved. Other QRDR mutations seem not to be related with quinolone resistance.
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Acknowledgements Chiara Seminati is supported by a fellowship of the Ministerio de Educacio´ n y Ciencia of Spain. This work was partially funded by project AGF99-1234 of the Spanish Ministerio de Educacio´ n y Ciencia.
References Allen, K.J., Poppe, C., 2002. Phenotypic and genotypic characterization of food animal isolates of Salmonella with reduced sensitivity to ciprofloxacin. Microb. Drug Resist. 8, 375–383. Baucheron, S., Tyler, S., Boyd, D., Mulvey, M.R., Chaslus-Dancla, E., Cloeckaert, A., 2004. AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar typhimurium DT104. Antimicrob. Agents Chemother. 48, 3729–3735. Deguchi, T., Yasuda, M., Nakano, M., Ozeki, S., Ezaki, T., Maeda, S., Saito, I., Kawada, Y., 1996. Rapid detection of point mutations of Neisseria gonorrhoeae gyrA gene associated with decreased susceptibilities to quinolones. J. Clin. Microbiol. 34, 2255–2258. Eaves, D.J., Randall, L., Gray, D.T., Buckley, A., Woodward, M.J., White, A.P., Piddock, L.J., 2004. Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica. Antimicrob. Agents Chemother. 48, 4012–4015. Everett, M.J., Jin, Y.F., Ricci, V., Piddock, L.J., 1996. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob. Agents Chemother. 40, 2380–2386. Ferrero, L., Cameron, B., Manse, B., Lagneaux, D., Crouzet, J., Famechon, A., Blanche, F., 1994. Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones. Mol. Microbiol. 13, 641–653. Friedman, S.M., Lu, T., Drlica, K., 2001. Mutation in the DNA gyrase A gene of Escherichia coli that expands the quinolone resistance-determining region. Antimicrob. Agents Chemother. 45, 2378–2380.
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Griggs, D.J., Gensberg, K., Piddock, L.J.V., 1996. Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals. Antimicrob. Agents Chemother. 40, 1009– 1013. Hooper, D.C., 2001a. Mechanisms of action of antimicrobials: focus on fluoroquinolones. Clin. Infect. Dis. 32 (Suppl 1), S9–S15. Hooper, D.C., 2001b. Emerging mechanisms of fluoroquinolone resistance. Emerg. Infect. Dis. 7, 337–341. Ling, J.M., Chan, E.W., Lam, A.W., Cheng, A.F., 2003. Mutations in topoisomerase genes of fluoroquinolone-resistant salmonel– lae in Hong Kong. Antimicrob. Agents Chemother. 47, 3567– 3573. Mateu, E.M., Martin, M., Darwich, L., Mejia, W., Frias, N., Garcia Pen˜ a, F.J., 2002. Antimicrobial susceptibility of Salmonella strains isolated from swine in Catalonia, Spain. Vet. Rec. 150, 147–150. Mejia, W., Zapata, D., De Frutos, C., Martin, M., Casal, J., Mateu, E., 2003. Antimicrobial susceptibility of Salmonella isolated pig carriers. In: Proceedings of the Fifth International Symposium on the Epidemiology and Control of Foodborne Pathogens in Pork, Crete, Greece. Mølbak, K., Lau, B.D., Moller, A.F., Munk, J.E., Engberg, J., Frydenndahl, K., Gernee, P., Munk, A., Wegener, H.C., 1999. An outbreak of multidrug resistant quinolone-resistant Salmonella enterica serotype Typhimurium DT104. N. Engl. J. Med. 341, 1420–1425. NCCLS, 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard, 2nd ed. NCCLS Document M31-A2. NCCLS, Wayne, PA, USA. ISBN 1-56238-461-9. Piddock, L.J.V., Ricci, V., McLaren, I., Griggs, D.J., 1998. Role of mutation in the gyrA end parC genes of nalidixic-acid-resistant Salmonella serotypes isolated from animals in the United Kingdom. J. Antimicrob. Chemother. 41, 635–641. Walker, R.A., Saunders, N., Lawson, A.J., Lindsay, E.A., Dassama, M., Ward, L.R., Woodward, M.J., Davies, R.H., Liebana, E., Threlfall, E.J., 2001. Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104 isolates. J. Clin. Microbiol. 39, 1443–1448.
Short communication
Mutations in the quinolone-resistance determining region (QRDR) of Salmonella strains isolated from pigs in Spain C. Seminati a,b,*, W. Mejia a, E. Mateu a,b, M. Martin a,b a
Departament de Sanitat i d’Anatomia Animals, Universitat Auto`noma de Barcelona (UAB), Facultat de Veterinaria, 08193 Bellaterra (Cerdanyola del Valle`s), Barcelona, Spain b Centre de Recerca en Sanitat Animal (CReSA), Universitat Auto`noma de Barcelona (UAB), 08193 Bellaterra (Cerdanyola del Valle`s), Barcelona, Spain Received 23 June 2004; received in revised form 9 December 2004; accepted 23 December 2004
Abstract Quinolone-resistance determining region (QRDR) of Gyrase A gene was sequenced in 54 Salmonella strains of pig origin that have different quinolone-resistance patterns. Those strains accounted for 12 different serotypes. Mutations at Ser83 or Asp87 were predominant in the studied isolates. However, for serotypes Anatum and Virchow, resistance to quinolones seemed to be linked to specific mutations, namely, Ser83 ! Tyr and Ser83 ! Phe, respectively. Other mutations found in different positions did not seem to have clinical significance except for changes at Asp82. # 2005 Elsevier B.V. All rights reserved. Keywords: Quinolone; Resistance; QRDR; Salmonella; Pig
1. Introduction Nowadays, an intense debate on the causes of the increasing antimicrobial resistance is being held among scientists. It has been suggested that the non-therapeutic use of antimicrobial agents in animals is one of the main factors that contribute to the emergence of drug resistances. Particularly, there is a concern for the development of resistant zoonotic bacteria such as Salmonella that may pass to people * Corresponding author. Tel.: +34 93 5811046; fax: +34 93 5813297. E-mail address: [email protected] (C. Seminati).
through the food chain and can produce untreatable disease. For example, a Salmonella strain of pig origin with decreased susceptibility to quinolones caused a severe outbreak of salmonellosis in Denmark where two people died (Mølbak et al., 1999). Fluoroquinolones have become the first choice treatment for septicemic salmonellosis and thus, the emergence of quinolone-resistant Salmonella strains causes a considerable concern. Acquired resistance to fluoroquinolones arises from several mechanisms: mutations of the target enzymes, modification of membrane porins or overproduction of proteins involved in efflux systems such as the AcrAB-TolC (Everett et al., 1996; Hooper,
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.12.024
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2001a, 2001b; Baucheron et al., 2004). In Gram negative bacteria, quinolone resistance is usually conferred by point mutations in target enzymes, especially those encoding for the DNA gyrase (gyrA, gyrB, parC, parE). For Salmonella, mutations in the gyrA gene, that encodes for the subunit A of the DNA gyrase, seem to be responsible for the development of most resistances to quinolones. These mutations have been clustered in a region of the gene product between amino acids 67 and 122, the so-called quinoloneresistance determining region (QRDR) (Deguchi et al., 1996; Ferrero et al., 1994). Single amino acid changes at Ser-83 (change by Phe, Tyr, or Ala) or at Asp-87 (change by Gly, Asn, or Tyr) are the most frequent (Griggs et al., 1996). However, some authors suggested that this QRDR can be larger and could start at position 51 (Friedman et al., 2001). In the present study, we investigated the mutations in the QRDR of Salmonella isolates of porcine origin in order to figure out their distribution and significance.
Virchow (5), Anatum (4), Brandenburg (4), Hadar (3), Enteritidis (3), and one strain each of serotypes Abony, Bredeney, Grumpensis and Choleraesuis. According to the available data, isolates were epidemiologically unrelated. Selected isolates were cultured overnight in BHI broth at 37 8C. Then, cultures were centrifuged and suspended in 1 ml of Rnase and Dnase free water (Ambion). DNA extraction was done by boiling for 10 min. Amplification and sequencing of the gyrA QRDR region was done according to a previously described PCR protocol (Walker et al., 2001) modified from Griggs et al. (1996). With this technique the amplicon contained a region between codons 37 and 151 of the gyrA gene. PCR products were then sequenced using an ABI 373 A Stretch sequencer. Predicted amino acid sequences were compared to a quinolone-susceptible reference strain LT2 (GenBank accession number AE008801)
3. Results 2. Materials and methods Fifty-four Salmonella strains were selected from a bacterial collection of pig isolates of Catalonia (Spain) obtained from 1998 to 2002. Selected strains were chosen representing the most frequent pig isolates in Spain for that period. Susceptibility profiles of these isolates have been described before (Mateu et al., 2002; Mejia et al., 2003). Briefly, strains were classified for quinolone resistance according to the results of Kirby–Bauer disk diffusion test (nalidixic acid, and ciprofloxacin) or by the minimal inhibitory concentration determined in a microdilution test (enrofloxacin). All susceptibility tests were done according to NCCLS recommendations (NCCLS, 2002). Fourteen strains were susceptible to all quinolones. The remaining 40 strains were resistant or had decreased susceptibility to 1 or more drugs. For nalidixic acid, 37 strains were resistant (inhibition diameter 13 mm) and 3 were classified as intermediate (14–18 mm). For enrofloxacin 3 strains were resistant (MIC > 2.0 mg/ml) and 31 were intermediate (MIC > 0.5 and 2.0 mg/ml); for ciprofloxacin only 2 strains were intermediate (16–20 mm) and all others were susceptible. Distribution of isolates by serotype was: Typhimurium (15), Tilburg (9), 4,5,12:i:- (7),
Mutations in the gyrA gene were found in 42 strains (77.7%). The most frequent mutation (n = 23) corresponded to codon at position 87 (Asp ! Asn, 21 strains; Asp ! Tyr, two strains), followed by changes at position 83 (n = 9) (Ser ! Phe, six strains; Ser ! Tyr, three strains). Changes Asp87 ! Tyr were found only in serotype Enteriditis (two isolates) while all Ser83 ! Tyr changes were found only in serotype Anatum (three isolates). Five of the six strains with a change Ser83 ! Phe belonged to serotype Virchow. In one case, the recently described mutation at position 82 (Asp ! Arg) was found in one isolate of serotype Tilburg (MIC for enrofloxacin = 1.3 mg/ ml) that also had a mutation in Asp87 ! Asn. None of the strains had a double Ser83/Asp87 mutation. We also found other mutations in the gyrA QRDR that were not described before. These mutations could be found alone or in association with other changes. Thus, 16 strains mutated in position 78 (His ! Asn or less commonly His ! Asp). Four strains had a Pro79 ! His mutation, three a Gly107 ! Cys change and two strains had mutations in position 64 (Lys ! Asn and Lys ! Ser). These changes were found in several serotypes regardless whether or not a given strain was susceptible or resistant to quinolones.
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Table 1 Serovars and susceptibility profiles against quinolones for Salmonella isolates of pig origin with mutations in QRDR codons Ser83 or Asp87 Serovar
Disk diffusion test Nal
Cip
Virchow1 Virchow Virchow Virchow Virchow Tilburg Anatum Anatum Anatum 4.5.12:i:Hadar Hadar Tilburg Tilburg2 Tilburg Tilburg3 Tilburg Tilburg4 Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Tilburg Enteritidis Enteritidis
R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S R R
S S S S S S S S S S S S I S S S S S I S S S S S S S S S S S S S
MIC (mg/ml), Enr 0.7 0.7 0.7 0.7 1.3 0.7 1.3 NT 1.3 1.3 0.7 0.7 2.3 1.3 1.3 2.3 2.3 1.3 1.3 1.3 0.7 1.3 1.3 0.7 1.3 1.3 0.7 0.3 1.3 0.3 NT 0.7
Mutations in QRDR of gyrA Ser83
Asp87
Phe Phe Phe Phe Phe Phe Tyr Tyr Tyr Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Tyr Tyr
His78
Pro79
Asn Asn
His
Asp
Asp
Asn
Asn
Asp Asn
His His
QRDR: quinolone-resistance determining region; MIC: minimal inhibitory concentration. Nal: nalidixic acid; Cip: ciprofloxacin; Enr: enrofloxacin; S: susceptible; R: resistant; I: intermediate. Isolates that had other mutations—superscript 1: His80 ! Arg, Gln106 ! Leu; superscript 2: Asp82 ! Arg; superscript 3: Lys64 ! Asn; superscript 4 Gly107 ! Cys.
Twelve isolates had no mutations in the gyrA gene, but 2 were intermediate to enrofloxacin (MIC = 1.3 mg/ ml) and 2 more were susceptible to enrofloxacin (MIC = 0.1 mg/ml) although resistant to nalidixic acid. These results are summarized in Tables 1 and 2.
4. Discussion In our study we focused on gyrA mutations of Salmonella strains of pig origin that had been classified as resistant to nalidixic acid. Thirty-two of the examined strains had mutations in the clinically
significant positions of gyrA Ser83 and Asp87 but none had a double Ser83/Asp87 mutation. Nevertheless, some of them were resistant to enrofloxacin (MIC > 2.0 mg/ml). This fact indicates either that this single gyrA mutation can confer a high level of resistance, or that additional mechanisms were involved, for example, mutations in gyrB, parC or parE genes. However, the involvement of parC, parE and gyrB mutations in the development of quinolone resistance for salmonellae is controversial and some authors (Ling et al., 2003) suggested that parC and parE mutations appear mainly after gyrA changes developed, played thus a secondary role. Recently,
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Table 2 Serovars and susceptibility profiles against quinolones for Salmonella isolates of pig origin with mutations in QRDR other than Ser83 or Asp87 or without mutations Serovar
Disk diffusion test 1
Bredeney 4.5.12:i:Brandenburg Brandenburg Brandenburg Anatum 4.5.12:i:Enteriditis Tilburg Typhimurium2 4.5.12:i:Brandenburg Typhimurium Typhimurium 4.5.12:i:4.5.12:i:Choleraesuis Typhimurium Hadar Abony Grumpensis 4.5.12:i:-
Nal
Cip
S R I I I S S S R S R R R R S S S S S S S S
S S S S S S S S S S S S S S S S S S S S S S
MIC (mg/ml), Enr 0.3 0.1 1.3 1.3 1.3 0.3 0.1 0.1 1.3 0.1 0.1 1.3 0.1 1.3 0.1 0.1 0.3 0.1 NT 0.1 0.3 0.1
Mutations in QRDR of gyrA His78
Gly107
Asn Asn Asn Asn Asn Asn Asn Asn
Cys
Pro79
Lys64 Ser
His Cys
QRDR: quinolone-resistance determining region; MIC: minimal inhibitory concentration. Nal: nalidixic acid; Cip: ciprofloxacin; Enr: enrofloxacin; S: susceptible; R: resistant; I: intermediate. Isolates that had other mutations: superscript 1: Gln106 ! His; superscript 2: Gly108 ! Cys.
Eaves et al. (2004) described that Salmonella enterica strains with double gyrA and parC mutations were more susceptible to ciprofloxacin than other similar strains having a single gyrA mutation suggesting some kind of compensatory mechanism. It is interesting to note that some amino acid substitutions were only found in certain serotypes. That was the case for Ser83 ! Tyr in Salmonella Anatum and Ser ! Phe in Salmonella Virchow. In previous studies (Griggs et al., 1996; Piddock et al., 1998), the change Ser83 ! Phe has been reported for animal isolates of Salmonella Enteritidis. However, other changes have been not associated with a given serotype. The isolates that we studied were epidemiologically unrelated and, for that reason, it would be tempting to suggest that, in pigs, a given gyrA mutation can be associated to a given serotype. Notwithstanding, a larger study would be required to confirm this observation.
We also observed point mutations not described before. The most frequent ones corresponded to positions Lys64, His78, Pro79 and Gly107. These mutations were found regardless of the quinolonesusceptibility profile and we think that they are not related to the development of resistance. Regarding the strains that did not show any mutation, some were resistant to either nalidixic acid or were intermediate to enrofloxacin, a result that further indicates the existence of additional resistance mechanisms. However, the involvement of gyrB or parC mutations could not be ruled out (Allen and Poppe, 2002). Our results showed that Ser83 and Asp87 mutations seem to be one of the main mechanisms for the development of quinolone resistance in Salmonella of pig origin, but other mechanisms could be involved. Other QRDR mutations seem not to be related with quinolone resistance.
C. Seminati et al. / Veterinary Microbiology 106 (2005) 297–301
Acknowledgements Chiara Seminati is supported by a fellowship of the Ministerio de Educacio´ n y Ciencia of Spain. This work was partially funded by project AGF99-1234 of the Spanish Ministerio de Educacio´ n y Ciencia.
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