Antimicrobial resistance in clinical isolates of Salmonella enterica

Advance Access published March 28, 2003

Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkg193

Antimicrobial resistance in clinical isolates of Salmonella enterica serotype Enteritidis: relationships between mutations conferring quinolone resistance, integrons, plasmids and genetic types Sara M. Soto1, M. Angeles González-Hevia2 and M. Carmen Mendoza1* 1Departamento

de Biología Funcional, Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, C/Julián Clavería 6, 33006 Oviedo; 2Laboratorio de Salud Pública, Consejería de Sanidad, Principado de Asturias, Carretera del Rubín s/n, 33001 Oviedo, Spain Received 2 January 2002; returned 25 May 2002, revised 22 August 2002; accepted 4 February 2003

Introduction Human salmonellosis caused by Salmonella enterica serotype Enteritidis has greatly increased worldwide over the past two decades.1–7 An additional problem has been the emergence and spread of strains exhibiting resistance to antimicrobial drugs, particularly nalidixic acid and ampicillin.2–7 Infections with strains resistant to these antimicrobials may be difficult to treat because both antimicrobials are used in first-line therapy in invasive illness. The most frequent quinolone resistance mechanism involves mutations in the gyr genes coding for DNA gyrase.8,9 Single mutations, usually in the gyrA gene, have been associated with a high level of resistance to nalidixic acid and decreased susceptibility to fluoroquinolones, and double mutations with a higher level of resistance to fluoroquinolones.8,9 The most frequent ampicillin resistance mechanism is the inactivation of the antimicrobial by β-lactamases, encoded by bla genes, which may be located on different plasmids or on the chromosome. Plasmids

may, in turn, carry integrons and/or transposons, which may also be carried on the chromosome. 2,4,6,10 The aim of the present work was to ascertain the situation regarding antimicrobial resistance in Salmonella Enteritidis isolated in the Principality of Asturias (PA), Spain, in 2002. Asturias is a Northern Spanish Region with ∼1 million inhabitants and covering an area of ∼10 565 km2. In the past, antimicrobial resistance has been rare in this serotype. However, in the early and late 1990s the occurrence of resistance to ampicillin and nalidixic acid has increased, whereas resistance to other antimicrobials remained infrequent.

Materials and methods A total of 481 isolates from clinical samples and four control strains were analysed (Table 1). The clinical isolates were from faeces (454), blood (12) and other sites (15); resistant isolates were associated with 72 outbreaks and 129 sporadic episodes.

..................................................................................................................................................................................................................................................................

*Correspondong author. Fax: +34-985103148; E-mail: [email protected] ...................................................................................................................................................................................................................................................................

Page 1 of 5 © 2003 The British Society for Antimicrobial Chemotherapy

Downloaded from http://jac.oxfordjournals.org/ by guest on June 1, 2013

In 481 clinical isolates of Salmonella enterica serotype Enteritidis collected from a Spanish region in 2000, 108, 83 and four isolates were resistant, respectively, to nalidixic acid, ampicillin or both. Nalidixic acid resistance was the result of DNA gyrase mutations involving the codons Asp-87 (97 isolates) and Ser-83 (15 isolates) of the gyrA gene; no mutations in parC were detected. In ampicillin-resistant strains, blaTEM genes located on plasmids and/or the chromosome were implicated. Five plasmids containing blaTEM1-like genes were identified, ranging from 7 to 100 kb, four of which were self-transferable; one of these contained a class 1 sul1 integron with an aadA1a gene cassette. This integron was also found on the chromosome of an isolate resistant to ampicillin, streptomycin and sulfadiazine. A relationship between a 40 kb selftransferable plasmid and strains of Salmonella Enteritidis phage type 6a with a distinctive RAPD profile was established.

S. M. Soto et al. Antimicrobial susceptibility was determined by disc diffusion, according to the NCCLS technique,11 using commercial discs (bioMérieux and Oxoid, Madrid, Spain). Escherichia coli ATCC 25922 was used as the control strain. The antimicrobials and concentrations in µg are shown in the footnotes to Table 1. MICs of nalidixic acid, ciprofloxacin and

norfloxacin were determined by an agar dilution method,11 using serial dilutions of nalidixic acid 0.4–256 mg/L (Sigma, Madrid, Spain), ciprofloxacin and norfloxacin 0.06–1 mg/L (Bayer AG, Leverkusen, Germany). β-Lactamases were extracted by sonication from ampicillin-resistant (AMPr) isolates, subjected to isoelectrofocusing (IEF) gel electro-

Table 1. Features of antimicrobial-resistant Salmonella serotype Enteritidis isolates and control strains No. of isolates (n = 199)

Resistancea (bla pattern)b

DNA gyrase mutations (n)

Ampicillin plasmidsc

Integron IR/ cassetted

RAPD type (n)

Phage type (PI/TI)

108

nalidixic acid

gyrA Ser-83 (12)

–

–

C1 (12)

PT1 (2/5), PT4 (2/5), PT21 (1/5)

gyrA Asp-87 (96)

–

–

gyrA Ser-83 (3)

–

–

C1 (94) C2 (2) C1 (2)

– – – – – –

C1 (1) C6 (1) C1 (3) C6 (61) C6 (1) C6 (3) C1 (1)

PT4 (1/1) PT6a (1/1)

PT6 (2/3), PT21 (1/3) PT6a (1/1)

4

1

6

1

2 6 Controlse ATCC13076 CPHL-PT4 LSP313/93 LSP473/98

PT1 (1/1) PT4 (2/2)

ampicillin (blaR1) sulfadiazine

–

pUO-SeR2 pUO-SeR1 – pUO-SeR1 pUO-SeR2 pUO-SeR1

ampicillin (blaR1) streptomycin/ spectinomycin sulfadiazine ampicillin (blaR2)

–

pUO-SeR3 pUO-SeR4

– 1000/aadA1a

C1 (3) C6 (1)

– pUO-SeR1

– –

pUO-SeR5 pUO-SeR3

– 1000/aadA1a

C1 (1) C6 (3) C1 (1) C1 (1) C1 (1)

– –

pUO-SeR1 –

– –

C6 (2) C1 (6)

PT6a (2/2)

– – – –

– – pUO-SeR1 pUO-SeR1

– – – 1000/aadA1a

C2 C1 C6 C6

PT1 PT4 PT6a PT6a

ampicillin (blaR1)

ampicillin (blaR2) streptomycin/ spectinomycin sulfadiazine ampicillin (blaR3) other – – ampicillin (blaR1) ampicillin (blaR1) streptomycin Sulfadiazine

gyrA Asp-87 (1) –

–

PT6a (8/8) PT6a (1/1) PT6a (2/2) PT4 (1/1)

PT6a (2/2) PT1 (1/1) PT4 (1/1)

A total of 481 isolates was examined, the 199 resistant isolates are shown here. aAntimicrobials and concentrations in µg tested: amikacin 30; ampicillin 10; ciprofloxacin 5; chloramphenicol 30; gentamicin 10; imipenem 10; kanamycin 30; streptomycin 10; nalidixic acid 30; tetracycline 30; sulfadiazine 300; and trimethoprim-sulfamethoxazole 1.25–23.75. In addition, the ampicillin-resistant isolates were also tested with co-amoxiclav 30; cefalothin 30; ceftazidime 30; cefotaxime 30; and piperacillin 100; the streptomycin-resistant isolates with spectomycin 30. bblaR1–R3, β-lactam resistance patterns; blaR1 (ampicillin), blaR2 (ampicillin/piperacillin) and blaR3 (ampicillin/cefalothin/cefotaxime). cpUO-SeR1-R5, plasmid of University of Oviedo Salmonella enteridis Resistance 1–5 (see Figure 1 and text). dIR, size in bp of the inserted region in class 1 integrons/gene cassette inserted. eControl strains: ATCC, American Type Culture Collection; CHPL, Central Health Public Laboratory (Colindale, UK); LSP, Laboratorio de Salud Pública (Principality of Asturias, Spain).

Page 2 of 5


71

nalidixic acid ampicillin (blaR1)

Antimicrobial resistance in Salmonella Enteritidis

Results Fifty-seven per cent of isolates were susceptible to all drugs, with 23%, 18%, 2% and 256 mg/L) coupled with decreased susceptibility to fluoroquinolones (MIC 0.25–2 mg/L), only mutations in the gyrA gene were detected (20% and 3% involved codons Asp-87 and Ser-83, respectively). All AMPr isolates contained blaTEM genes on the chromosome and/or on plasmids. Five types of AMPr plasmids were defined by their molecular mass, restriction profile and transferability. A self-transferable 40 kb plasmid (pUO-SeR1) was the most frequent. In another study conducted in Greece,2 ampicillin resistance was also mainly the result of the spread of a limited number of clones of PT6a, carrying related 54 kb self-transferable plasmids containing bla-TEM1.2 EcoRI restriction profiles of AMPr plasmids from Greece and Asturias were compared, revealing some similarities. AMPr plasmids differing in mass, resistance pattern,

restriction profile and incompatibility group have been described in Enteritidis collected in different countries,2,4,5,16 and it is noteworthy that some AMPr plasmids are capable of converting strains belonging to PTs 1 and 4 to PT6a, and PT8 to PT13.15 In contrast, only limited work on integrons in Salmonella Enteritidis has been reported;4,6,10 in these studies only the integron-associated aadA1a gene cassette has been identified. In this investigation, RAPD analysis was applied to differentiate Enteritidis isolates, together with R pattern and plasmid analysis. RAPD is simple, reproducible, screens stable traits and shows good discriminatory power within and between Salmonella serotypes.14 The results have demonstrated that, in PA, ampicillin resistance in Salmonella Enteritidis is mediated mostly by pUO-SeR1 plasmids and associated mainly

Page 4 of 5


Figure 1. Analysis of plasmids from Enteritidis isolates. (a) Plasmid profiles. (b) Hybridization of (a) with spvC (A), blaTEM1 (B), aadA1a (C) and qacE∆1-sul1 (D) probes. Lanes P and T: standards of plasmid size (P, of 60, 5.6, 4.8, 3.5, 3.3 kb; and T, of 90 and 2.8 kb). Lanes 1–11: plasmid profiles generated by Enteritidis clinical isolates. (c and e) Plasmid restriction profiles generated with EcoRI and ClaI, respectively. (d and f) Hybridization of (c) and (d) with blaTEM1 (B), aadA1a (C) and qacE∆1-sul1 (D) probes. Lanes P1 and P2: plasmid profiles of Enteritidis LSP313/93 and LSP49/00, respectively, used as controls. Lanes R1 to R4: representing pUO-SeR1 to pUO-SeR4 plasmids from different isolates.

Antimicrobial resistance in Salmonella Enteritidis with a type identified as PT 6a, RAPD-C6. In a minority of strains, other types of AMPr plasmid (pUO-SeR2-R5) have also been identified, but for the most part in isolates obtained after 1997.

6. Guerra, B., Soto, S. M., Cal, S. & Mendoza, M. C. (2000). Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes. Antimicrobial Agents and Chemotherapy 44, 2166–9.

Acknowledgements

7. Cruchaga, S., Echeitia, A., Aladueña, A., Garcia-Peña, J., Frias, N. & Usera, M. A. (2001). Antimicrobial resistance in salmonellae from humans, food and animals in Spain in 1998. Journal of Antimicrobial Chemotherapy 47, 315–21.

References 1. Rodrigue, D. C., Tauxe, R. V. & Rowe, B. (1990). International increase in Salmonella enteritidis: a new pandemic. Epidemiology and Infection 105, 21–7. 2. Tassios, P. T., Markogiannakis, A., Vatopoulos, A. C., Katsanikou, E., Velonakis, E. N., Kourea-Kremastinou, J. et al. (1997). Molecular epidemiology of antibiotic resistance of Salmonella enteritidis during a 7-year period in Greece. Journal of Clinical Microbiology 35, 1316–21. 3. Centro Nacional de Epidemiología. (2000). Resultados de las principales identificaciones bacterianas declaradas al sistema de información microbiológica en la semana 52 que terminó el 30 de diciembre de 2000. Boletin Epidemiológico Semanal 8, 274. 4. Nastasi, A., Mammina, C. & Cannova, L. (2000). Antimicrobial resistance in Salmonella Enteritidis, Southern Italy, 1990–1998. Emerging Infectious Diseases 6, 401–3. 5. Ling, J. M., Koo, I. C., Kam, K. M. & Cheng, A. F. (1998). Antimicrobial susceptibilities and molecular epidemiology of Salmonella enterica serotype Enteritidis isolated in Hong Kong from 1986 to 1996. Journal of Clinical Microbiology 36, 1693–9.

8. Vila, J., Ruíz, J., Marco, F., Barcelo, A., Goñi, P., Giralt, E. et al. (1994). Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrobial Agents and Chemotherapy 38, 2477–9. 9. Giraud, E., Brisabois, A., Martel, J. & Chaslus-Dancla, E. (1999). Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field. Antimicrobial Agents and Chemotherapy 43, 2131–7. 10. Brown, A. W., Rankin, S. C. & Platt, D. J. (2000). Detection and characterization of integrons in Salmonella enterica serotype Enteritidis. FEMS Microbiology Letters 191, 145–9. 11. National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing— Ninth Informational Supplement: Approved Standard M100-S9. NCCLS, Wayne, PA, USA. 12. Ward, L. R., de Sa, J. D. & Rowe, B. (1987). A phage-typing scheme for Salmonella enteritidis. Epidemiology and Infection 99, 291–4. 13. Guerra, B., Soto, S. M., Argüelles, J. M. & Mendoza, M. C. (2001). Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:-]. Antimicrobial Agents and Chemotherapy 45, 1305–8. 14. Soto, S. M., Guerra, B., González-Hevia, M. A. & Mendoza, M. C. (1999). Potential of a three-way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes. Applied and Environmental Microbiology 65, 4830–6. 15. Sambrook, J., Fritch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. 16. Ridley, A. M., Punia, P., Ward, L. R., Rowe, B. & Threlfall, E. J. (1996). Plasmid characterization and pulsed-field electrophoretic analysis demonstrate that ampicillin-resistant strains of Salmonella enteritidis phage type 6a are derived from Salmonella enteritidis phage type 4. Journal of Applied Bacteriology 81, 613–8.

Page 5 of 5


We thank M. A. Usera and Ana Aladueña of CNM for phage typing Enteritidis isolates; J. Vila and M. Navia of the ‘Hospital Clínico de Barcelona’ for their help in β-lactamase characterization; the personnel of the Microbiology Laboratories of the ‘Hospital Central de Asturias’ (Oviedo), ‘Hospital San Agustín’ (Avilés), ‘Hospital de Jarrio’, ‘Hospital de Cabueñes’ (Gijón) and ‘Hospital Carmen and Severo Ochoa’ (Cangas del Narcea) for their invaluable collaboration with the LSP in registering clinical isolates of Salmonella. We particularly wish to thank Beatriz Guerra for her help and advice in the experimental phase of this work. This work has been supported by a grant from the ‘Fondo de Investigación Sanitaria’ (Ref. 00/1084). S.M.S. is the recipient of a grant from the ‘Formación de Profesorado Universitario’ (Ref. AP98) of the Spanish Ministry of Culture and Education.