1 Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 12, 2781901 Oeiras; 2 Estação Agronómica Nacional, Instituto Nacional de Investigação Agrária, 2781505 Oeiras; 3 Faculdade de Ciências/Departamento de Biologia Vegetal/Centro de Genética e Biologia Molecular, Universidade de Lisboa, 1749016 Lisboa, Portugal
Received 4 February 2003; returned 2 March 2003; revised 17 March 2003; accepted 27 April 2003
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Abstract |
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Keywords: aminoglycosides, antibiotic resistance, enterococci
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Introduction |
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One of the antibiotics of major concern is gentamicin.2 Severe enterococcal infections are treated using a combination of a cell-wall active agent and an aminoglycoside, typically gentamicin, which replaced streptomycin after high-level resistance to the latter was reported in the late 1970s.3,4 However, in the 1980s, the first cases of high-level gentamicin resistant (HLGR) enterococci were reported.5 The appearance of an increasing number of HLGR enterococci in the clinical environment makes it difficult to treat severe cases of endocarditis because synergism with cell-wall active agents (such as ampicillin, penicillin G or vancomycin) no longer works.6 Moreover, HLGR enterococci are no longer limited to the clinical setting, and can be found in a variety of aquatic environments2 and food products, namely from animal origin.7,8 In fact, of the more than 1 million tons of antibiotics released into the biosphere during the last 50 years, 50% are estimated to come from veterinary and agricultural settings, and aminoglycosides are among these antibiotics.9
HLG resistance in enterococci is defined by MICs of >2000 mg/L.5 However, enterococci isolates with MICs as low as 500 mg/L are also considered HLGR,10,11 and some authors even estimate that isolates with MICs > 128 mg/L exhibit such resistance.4 In Gram-positive cocci, high-level aminoglycoside resistance is generally due to enzymes that modify the antibiotic.4 The reactions catalysed by these enzymes can be divided into three categories: phosphorylation, adenylation and acetylation. The spread of HLG resistance is mainly because the genes that are responsible for it can be transferred through plasmids4,12 and transposons,3,13 and thus potentially may disseminate among enterococci.
It is important to ascertain whether the large number of enterococci in milk and cheese constitute any threat in terms of HLG resistance. Therefore, gentamicin resistance was screened for in enterococci isolated from Portuguese dairy products. For comparison, clinical strains, both from hospitals and veterinary settings, were also studied, as were type strains for all the species of the genus Enterococcus. Three phosphorylase genes and a bifunctional enzyme were screened for. These were, respectively, aph(2'')-Ib, which confers HLG resistance,14 aph(2'')-Ic, which confers mid-level gentamicin resistance,15 and aph(2'')-Id, which confers HLG resistance;16 the bifunctional enzyme aac(6')-Ie-aph(2'')-Ia also confers HLG resistance.17
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Materials and methods |
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A total of 90 enterococci were used in this study: 50 isolated from Portuguese dairy products, from the IBET culture collection; 11 isolated from dog infections (provided by Constança Feria, of the Medical Veterinary Faculty of Lisbon Technical University); and 29 isolated from human infections in hospitals (provided by Aida Duarte, of the Faculty of Pharmacy at Lisbon University, and Manuela Caniça, at the National Institute of Health Dr Ricardo Jorge). The dairy isolates were identified by P. I. Alves, M. P. Martins, T. Semedo, J. J. F. Marques, R. Tenreiro & M. T. B. Crespo (results not shown) as 17 Enterococcus faecalis, four Enterococcus faecium, 21 Enterococcus durans, one Enterococcus raffinosus, six Enterococcus hirae and one Enterococcus spp. Among the 40 clinical isolates, 26 were identified as E. faecalis, seven as E. faecium, one as Enterococcus solitarius and six as Enterococcus spp. E. faecalis DSMZ 12956, E. faecium SF 11770 (provided by J. W. Chow, Wayne State University School of Medicine, Detroit, MI, USA), Enterococcus gallinarum SF 9117 (provided by D. B. Clewell, University of Michigan, Ann Arbor, MI, USA) and Enterococcus casseliflavus UC 73 (provided by J. W. Chow) were also used in the present work, both in the antibiotic susceptibility assays and in PCR as positive controls for the aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib, aph(2'')-Ic and aph(2'')-Id genes, respectively. Twenty-six enterococci reference strains obtained from DSMZ, CECT (Colección Española de Cepas Tipo, Valencia, Spain), LMG (Laboratorium voor Microbiologie, Gent, Belgium) and ATCC were also used (see Table 3). Enterococcus pallens and Enterococcus gilvus were not included in this study since they were proposed and accepted as new species only recently.18
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Susceptibility to gentamicin was determined using the NCCLS disc diffusion method.19 Disc diffusion zone diameters were determined on MuellerHinton agar (Oxoid, UK) and measured using a calliper. Two gentamicin disc concentrations were tested (10 and 120 µg) (Oxoid), and strains were classified as resistant, intermediate and susceptible according to bioMérieux20 (for the 10 µg discs) and the NCCLS21 (for the 120 µg discs). Staphylococcus aureus ATCC 25923 was used as the positive control for the 10 µg discs, and E. faecalis ATCC 29212 for quality control of the 120 µg discs, as recommended by the NCCLS.21
MIC determination
MIC95 for gentamicin was determined using two different methods: the macrodilution method, as described by the NCCLS;22 and the Etest (Biodisk, Sweden), used according to the manual. For the macrodilution method, an inoculum of 105 to 106 cfu/mL was chosen. Gentamicin was purchased from Sigma (Germany) and concentrations used were 0.125, 2, 4, 8, 16, 32, 64, 128 and 512 mg/L. E. faecalis ATCC 29212 was used as the control for the dilution method and E. faecalis ATCC 51299 as the control for the Etest.
Preparation of DNA
Total DNA was extracted from cells according to the method of Pitcher et al.23 Plasmid DNA was extracted using the method of Anderson & McKay,24 with the following modifications: incubation with lysozyme lasted for 30 min and with double the concentration of the enzyme.
PCR and DNA sequencing
PCR was performed with a T-personal Combi thermocycler (Biometra, Göttingen, Germany). Each 50 µL PCR mixture contained 250 ng of DNA, 0.5 µM of each primer, 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP), 1 ¥ PCR buffer, 0.005% W1 and 2.5 U of Taq DNA polymerase. All reagents were purchased from GibcoBRL (Life Technologies, UK), except for the primers, which were purchased from MWG-Biotech (Germany). For amplification of the aac(6')-Ie-aph(2'')-Ia gene, the thermocycler was programmed with the following conditions: 5 min at 95°C, 30 s at 94°C, 30 s at 47°C and 30 s at 72°C for 30 cycles; 10 min at 72°C; and 4°C until analysis. For amplification of aph(2'')-Ib, aph(2'')-Ic and aph(2'')-Id, the thermocycler was programmed with the following conditions: 3 min at 95°C; 1 min at 95°C, 50 s at 57°C and 40 s at 72°C for 30 cycles; 5 min at 72°C; and 4°C until analysis. The primers used to detect the aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib, aph(2'')-Ic and aph(2'')-Id genes are described in Table 1. Visualization of amplicons was conducted with ethidium bromide (Sigma, Germany) under UV irradiation, after electrophoresis on 2% agarose (GibcoBRL, Germany) gels. Image analysis was performed with Kodak Digital Science (Germany). Amplification of the genes was confirmed with sequencing of the PCR products, after their purification either with Concert Rapid PCR Purification System or with Concert Matrix Gel Extraction System, both purchased from GibcoBRL (Germany). Sequencing was performed by STAB Vida Lda (Portugal).
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Results and discussion |
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The results do not agree with the generalized idea that enterococci are intrinsically resistant to gentamicin. In fact, for the 10 µg gentamicin disc, 42% of dairy isolates were susceptible, 71% of which had MICs £ 4 mg/L (Table 2, Etest). Among the 58% of resistant dairy isolates, 93% presented MICs > 4 mg/L (Etest) (between 8 and 32 mg/L). All dairy isolates (but one) presented MICs £ 32 mg/L, which is considerably inferior to the MICs generally accepted as low-level resistant for some authors, but is in accordance with others, as discussed above. Based on these results, we propose that MICs for LLR generally should be considered > 4 mg/L, and specifically between 8 and 32 mg/L.
If 4 mg/L is considered as the threshold value for low-level gentamicin resistance, the presented results show that the clinical isolates studied are intrinsically resistant to gentamicin. In fact, all clinical isolates (both human and veterinary) presented MICs >4 mg/L (Etest, Table 2) and, except for five isolates for which MICs were 1024 mg/L (Table 4), and one with an MIC of 128 mg/L (Table 4), 83% of the isolates presented MICs between 8 and 64 mg/L (Table 2). Moreover, only 7.5% of the clinical isolates behaved as susceptible with the 10 µg disc. These results show that dairy enterococcal isolates still represent a different scenario from clinical isolates and, therefore, should be included in future generalizations on antibiotic susceptibility of enterococci. In fact, previous work has shown that enterococci isolated from dairy products are susceptible to gentamicin.1
The reference strains, which represent the genus Enterococcus, behaved similarly to the dairy isolates, showing 58% resistant strains with the 10 µg disc (Table 2). All reference strains presented MICs < 32 mg/L, and nine <4 mg/L (Table 3). From these nine isolates, only one, Enterococcus dispar, was isolated from a clinical setting; all the others came from chickens, humans, donkeys, pigeons or milk. E. faecium and E. faecalis both presented MICs corresponding to low-level gentamicin resistance (Table 3).
None of the dairy enterococcal isolates showed HLG resistance, as determined by disc diffusion with gentamicin 120 µg, which is in contrast to recent reports, of nearly 14% HLGR isolates from food.7 The incidence of gentamicin resistance is generally low in Europe, but some strains isolated from meat have been shown to be HLGR,8 indicating that isolates exhibiting such resistance are not limited to the clinical setting, and should be monitored constantly for this resistance. Only 5% (two isolates) of the clinical isolates showed this characteristic when the disc diffusion susceptibility test was used with 120 µg gentamicin discs, and 95% behaved as susceptible with the 120 µg disc (Table 4). Only one reference strain, Enterococcus ratti, behaved as resistant with the high concentration gentamicin disc, and only E. faecalis CECT 187 behaved as intermediate with the same gentamicin disc.
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Table 4 shows that the bifunctional gene, aac(6')-Ie-aph(2'')-Ia, which encodes for HLG resistance, was detected in E. faecalis isolates and Enterococcus spp., but only in the hospital and veterinary environment, although previous work has been able to detect it in E. faecalis and E. faecium food isolates.7 However, homologous forms of this gene have been described in other species, namely Enterococcus avium, E. gallinarum, E. raffinosus and E. casseliflavus.6 The presence of the bifunctional gene does in fact confer HLG resistance, as shown by results in Table 4, which is in accordance with previous work.10,27 But such resistance is not always detected by the 120 µg gentamicin disc, as in the case of isolates 5HSJ, 344/99 and DSMZ 12956 (Table 4). Whatever the species, whether E. faecalis, E. faecium, E. durans, E. raffinosus or E. hirae, the bifunctional gene was not detected in any of the dairy isolates studied in this work.
Only one enterococcus, among 50 dairy isolates, gave positive amplification of the gene aph(2'')-Ib (isolate LN 9), which is described as conferring HLG resistance.14 LN 9 presented an MIC of 1.5 mg/L. Similar behaviour occurred with the clinical isolate HSM 3143a, with which aph(2'')-Ic was detected. This gene is described as conferring mid-level resistance to gentamicin,16 with MICs as low as 128 mg/L, using the Etest, and between 256 and 512 mg/L, using the dilution-in-tube test.10 However, HSM 3143a presented an MIC as low as 32 mg/L. Similar results, showing a discrepancy between high- and mid-level gentamicin resistance genes and the presented MICs were found in the report by Chow et al.10 with enterococcal clinical isolates. Moreover, the presence of aph(2'')-Ic in strain SF 9117 did confer HLG resistance, although according to Chow et al.15 the expected MIC for this strain was 256 mg/L. Thus, there seems to be no correlation between the presence of one of these genes and phenotypical behaviour of resistance. One hypothesis is that the detected genes may not be intact, or may be silent. This is a question for further analysis.
The gene aph(2'')-Id, which confers HLG resistance,16 was not found in any of the dairy or clinical isolates tested in this work.
Two clinical isolates, HSM 4182 and 31 rot, for which HLG resistance was detected by MIC determination, did not amplify any of the genes tested in the present work. However, other genes have been described that do not confer HLG resistance.31 These isolates are good candidates for as yet undescribed genes conferring such resistance.
The concomitant presence of two or more genes did not occur in any of the enterococcal isolates used in this work.
Overall, the results from this work show clearly that dairy enterococci are not intrinsically resistant to gentamicin, as generally accepted. This is in contrast to clinical isolates, where gentamicin resistance does appear to be intrinsic. Moreover, dairy isolates did not show HLG resistance, despite the fact that the gene aph(2'')-Ib was found in one of the isolates. This result suggests the possibility of gene transfer, probably from clinical or commensal bacteria to dairy enterococci, and may become a problem in the future. Therefore, gentamicin resistance should be monitored in dairy and in environmental isolates in general. Clinical isolates from Portuguese hospitals and veterinary settings behaved as expected from the literature published on clinical enterococcal isolates, both concerning HLG resistance and the presence of the bifunctional gene aac(6')-Ie-aph(2'')-Ia. The latter eliminates synergistic killing between aminoglycosides and cell-wall active antibiotics. The disc diffusion method for the detection of HLG resistance, despite being easy to handle, should be substituted by MIC determination, which has been shown to be more reliable.
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Acknowledgements |
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This work was supported by Fundação para a Ciência e Tecnologia through Project POCTI/AGR/39371/2001. Maria de Fátima Silva Lopes thanks Fundação para a Ciência e Tecnologia for the grant PRAXIS XXI BPD/22122/99.
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Footnotes |
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References |
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2 . Rice, E. W., Messer, J. W., Johnson, C. H. et al. (1995). Occurrence of high-level aminoglycoside resistance in environmental isolates of enterococci. Applied and Environmental Microbiology 61, 3746.[Abstract]
3
.
Simjee, S., Fraise, A. P. & Gill, M. J. (1999). Plasmid heterogeneity and identification of a Tn5281-like element in clinical isolates of high-level gentamicin-resistant Enterococcus faecium isolated in the UK. Journal of Antimicrobial Chemotherapy 43, 62535.
4 . Patterson, J. E., Masecar, B. L., Kauffman, C. A. et al. (1988). Gentamicin resistance plasmids of enterococci from diverse geographic areas are heterogeneous. Journal of Infectious Diseases 158, 21216.[ISI][Medline]
5 . Maderski-Samoraj, B. & Murray, B. E. (1983). High-level resistance to gentamicin in clinical isolates of enterococci. Journal of Infectious Diseases 147, 7517.[ISI][Medline]
6 . Sahm, D. F. & Gilmore, M. S. (1995). High-level gentamicin resistance among enterococci. Developments in Biological Standardization 85, 99105.[Medline]
7
.
Franz, C. M. A. P., Muscholl-Silberhorn, A. B., Yousif, N. M. K. et al. (2001). Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Applied and Environmental Microbiology 67, 43859.
8 . Teuber, M., Meile, L. & Schwarz, F. (1999). Acquired antibiotic resistance in lactic acid bacteria from food. Antonie van Leeuwenhoek 76, 11537.[CrossRef][ISI][Medline]
9 . Teuber, M. (2001). Veterinary use and antibiotic resistance. Current Opinion in Microbiology 4, 4939.[CrossRef][ISI][Medline]
10 . Chow, J. W., Donabedian, S. M., Clewell, D. B. et al. (1998). In vitro susceptibility and molecular analysis of gentamicin-resistant enterococci. Diagnostic Microbiology and Infectious Diseases 32, 1416.[CrossRef][ISI][Medline]
11 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing. Eleventh Informational Supplement. MIC Testing. M100-S11. NCCLS, Villanova, PA, USA.
12 . Thal, L. A., Chow, J. W., Patterson, J. E. et al. (1993). Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance. Antimicrobial Agents and Chemotherapy 37, 1347.[Abstract]
13 . Thal, L. A., Chow, J. W., Clewell, D. B. et al. (1994). Tn924, a chromosome-borne transposon encoding high-level gentamicin resistance in Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 38, 11526.[Abstract]
14
.
Kao, S. J., You, I., Clewell, D. B. et al. (2000). Detection of the high-level aminoglycoside resistance gene aph(2'')-Ib in Enterococcus faecium. Antimicrobial Agents and Chemotherapy 44, 28769.
15 . Chow, J. W., Zervos, M. J., Lerner, S. A. et al. (1997). A novel gentamicin resistance gene in Enterococcus. Antimicrobial Agents and Chemotherapy 41, 51114.[Abstract]
16
.
Tsai, S. F., Zervos, M. J., Clewell, D. B. et al. (1998). A new high-level gentamicin resistance gene, aph(2'')-Id, in Enterococcus spp. Antimicrobial Agents and Chemotherapy 42, 122932.
17 . Ferretti, J. J., Gilmore, K. S. & Courvalin, P. (1986). Nucleotide sequence analysis of the gene specifying the bifunctional 6'-aminoglycoside acetyltransferase 2''-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities. Journal of Bacteriology 167, 6318.[ISI][Medline]
18
.
Tyrrell, G. J. Turnbull, L., Teixeira, L. M. et al. (2002). Enterococcus gilvus sp. nov and Enterococcus pallens sp. nov. isolated from human clinical specimens. Journal of Clinical Microbiology 40, 11405.
19 . National Committee for Clinical Laboratory Standards. (1993). Performance Standards for Antimicrobial Disk SusceptibilityTestsFifth Edition: Approved Standard M2-A5. NCCLS, Villanova, PA, USA.
20 . bioMérieux. (1996). Livret technique: Identification et antibiogramme. Methodes Manuelles, pp. 2223. bioMérieux, Marcy lÉtoile, France.
21 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing. Eleventh Informational Supplement. Disk Diffusion. M100-S11. NCCLS, Villanova, PA, USA.
22 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyThird Edition: Approved Standard M7-A3. NCCLS, Villanova, PA, USA.
23 . Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidinum thiocyanate. Letters in Applied Microbiology 8, 1516.[ISI]
24 . Anderson, D. G. & McKay, L. L. (1983). Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Applied and Environmental Microbiology 46, 54952.[ISI][Medline]
25 . Comité de lantibiogramme de la Société Française de Microbiologie. (1996). Zone sizes and MIC breakpoints for non-fastidious organisms. Clinical Microbiology and Infection 2, S46-9.[Medline]
26 . Murray, B. E. (1990). The life and times of the enterococcus. Clinical Microbiology Reviews 3, 4665.[ISI][Medline]
27 . Ounissi, H., Derlot, E., Carlier, C. et al. (1990). Gene homogeneity for aminoglycoside-modifying enzymes in Gram-positive cocci. Antimicrobial Agents and Chemotherapy 34, 21648.[ISI][Medline]
28 . Sahm, D. F. & Torres, C. (1988). High-content aminoglycoside disks for determining aminoglycoside-penicillin synergy against Enterococcus faecalis. Journal of Clinical Microbiology 26, 25760.[ISI][Medline]
29 . Swenson, J. M., Ferraro, M. J., Sahm, D. F. et al. (1995). Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. Journal of Clinical Microbiology 33, 300818.[Abstract]
30 . Sahm, D. F., Boonlayangoor, S., Iwen, P. C. et al. (1991). Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis. Journal of Clinical Microbiology 29, 19349.[ISI][Medline]
31 . Kak, V. & Chow, J. W. (2002). Acquired antibiotic resistances in enterococci. In The Enterococci: Pathogenesis, Molecular Biology and Antibiotic Resistance (Gilmore, M. S., Ed.), pp. 35583. ASM Press, Washington, DC, USA.
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