Gentamicin resistance in dairy and clinical enterococcal isolates and in reference strains

Maria de Fátima Silva Lopes1,*, Tânia Ribeiro1, Maria Paula Martins2, Rogério Tenreiro3 and Maria Teresa Barreto Crespo1

1 Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 12, 2781–901 Oeiras; 2 Estação Agronómica Nacional, Instituto Nacional de Investigação Agrária, 2781–505 Oeiras; 3 Faculdade de Ciências/Departamento de Biologia Vegetal/Centro de Genética e Biologia Molecular, Universidade de Lisboa, 1749–016 Lisboa, Portugal

Received 4 February 2003; returned 2 March 2003; revised 17 March 2003; accepted 27 April 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Enterococci isolated from Portuguese dairy products (milk and cheese) and clinical settings (hospitals and veterinary clinics), together with reference strains from the genus Enterococcus, were screened for low- and high-level gentamicin resistance using the standard disc diffusion method (10 and 120 µg gentamicin discs). MICs were also determined using both the macrodilution method and the Etest. Four genes [aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib, aph(2'')-Ic and aph(2'')-Id] responsible for high- and mid-level gentamicin resistance were sought using PCR. Although enterococci generally are regarded as being intrinsically resistant to low levels of gentamicin, results revealed that many dairy enterococci (around 30% of the isolates used) are not intrinsically resistant to gentamicin, showing MICs of £4 mg/L. High-level gentamicin resistance was not detected in any of the dairy isolates studied, except for aph(2'')-Ib, which was found in one. Therefore, gentamicin resistance should be monitored in dairy enterococci, although it does not seem to be a problem at present. In contrast, all clinical isolates studied were, as expected, intrinsically resistant to low levels of gentamicin, presenting MICs > 8 mg/L. Fifteen percent of these clinical isolates showed high-level gentamicin resistance (MICs > 512 mg/L), with the bifunctional gene aac(6')-aph(2'') being detected in four of them. However, discs with gentamicin 120 µg failed to detect some isolates with high-level gentamicin resistance.

Keywords: aminoglycosides, antibiotic resistance, enterococci


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Enterococci are ubiquitous bacteria that occur frequently in large numbers in dairy and other food.1 Along with ~450 other taxa of anaerobic and aerobic bacteria, Enterococcus are part of the normal intestinal microbiota. However, in recent years the prevalence of enterococci among nosocomial pathogens has increased, mostly as a result of their acquisition of multiple antimicrobial resistance.

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


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Microorganisms

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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Table 3.  Gentamicin MICs for the reference strains of the genus Enterococcus
 
Antibiotic susceptibility testing

Susceptibility to gentamicin was determined using the NCCLS disc diffusion method.19 Disc diffusion zone diameters were determined on Mueller–Hinton 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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Table 1.  Forward (f) and reverse (r) primers used in the PCR assay to amplify the genes coding for aminoglycoside-modifying enzymes studied in this work
 

    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Due to the increasing number of enterococcal clinical isolates showing HLGR, the present work aimed to understand the status of this characteristic in dairy enterococcal isolates and its comparison with hospital and veterinary isolates and with reference strains. Enterococci are considered intrinsically resistant to low-levels of aminoglycosides in general,2 and to gentamicin in particular. Therefore, criteria for classifying enterococcal isolates as resistant or susceptible to gentamicin, namely the NCCLS21 and CA-SFM,25 include only HLG resistance, and recommend the use of high content discs, with gentamicin 120 µg. However, there is no consensus on MIC criteria for low-level resistance (LLR). In fact, in the literature, different definitions for this resistance can be found: 2–8,26 2–16,2 8–32,4 <12827 and 64–128 mg/L.10 It is thus not easy to classify isolates from the environment, such as dairy isolates, as resistant or susceptible. In order to do so, and taking into consideration the comments above, gentamicin discs with 10 and 120 µg were used in this work. The results of the disc susceptibility test and MIC determination are presented in Table 2.


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Table 2.  Number of isolates, of dairy and clinical origin and reference strains, which are resistant (R), intermediate (I) and susceptible (S) (disc diffusion) and have MICs in the intervals represented, using two different methods: macrodilution and Etest
 
Results of determination of MICs, obtained using both the macrodilution method and the Etest, generally agree (Table 2). Therefore, either of these methods can be used when testing enterococci.

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.  Enterococcal isolates that had one of the genes that were screened for, showed resistance towards the 120 µg gentamicin disc or had MICs > 64 mg/L
 
The disc diffusion method, using a 120 µg disc, is considered a reliable detector of HLG resistance.2,26,28,29 However, when MICs > 500 mg/L are considered, the percentage of HLGR clinical isolates increases to 15%. This demonstrates the poor sensitivity of the 120 µg disc diffusion test in the detection of HLG resistance. In fact, among nine enterococci that presented MICs >= 512 mg/L, only four behaved as resistant with the 120 µg gentamicin disc (Table 4). False susceptibility, and not false resistance, is therefore a problem with this method, as reported in other work.30 However, E. ratti presented an MIC of 16 mg/L, but behaved as resistant with the 120 µg disc.

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.


    Acknowledgements
 
We thank Aida Duarte (University of Lisbon, Pharmacy Faculty), Constança Feria (Technical University of Lisbon, Medical Veterinary Faculty) and Manuela Caniça (National Institute of Health Dr Ricardo Jorge) for supplying clinical isolates. We are also grateful to D. B. Clewell (University of Michigan, USA) and J. W. Chow (Wayne State University School of Medicine, USA) for providing clinical and reference enterococcal strains.

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.


    Footnotes
 
* Corresponding author. Tel: +351-21-446-9551/2; Fax: +351-21-442-1161; E-mail: flopes{at}itqb.unl.pt Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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