The genetic background for streptomycin resistance in Escherichia coli influences the distribution of MICs

Marianne Sunde1,* and Madelaine Norström2

1 Section of Bacteriology and 2 Norwegian Zoonosis Centre, National Veterinary Institute, 0033 Oslo, Norway


*Corresponding author. Tel: +47-23-21-63-81; Fax: +47-23-21-63-01; Email: marianne.sunde{at}vetinst.no

Received 15 February 2005; returned 14 March 2005; revised 31 March 2005; accepted 6 April 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to investigate the genetic background for streptomycin resistance in Escherichia coli and perform analysis of the MICs in relation to genetic background.

Methods: The 136 strains investigated, with streptomycin MICs of ≥16 mg/L, originated from meat and meat products and were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET). PCR was carried out for detection of the streptomycin resistance genes strA-strB and the integron-associated aadA gene cassettes.

Results: The strA-strB genes and/or an aadA gene cassette were detected in 110 of the 136 (80.9%) strains investigated. The strA-strB genes were the most prevalent, and were detected in 90 strains. The aadA gene cassettes were detected in 29 strains, and nine strains harboured both the strA-strB genes and an aadA gene cassette. The distribution of MICs differed considerably between isolates harbouring the strA-strB genes (solely) (MIC50=128 mg/L) and isolates harbouring an aadA gene cassette (solely) (MIC50=16 mg/L). Strains harbouring both the strA-strB genes and an aadA gene cassette had higher streptomycin MICs than those harbouring either alone.

Conclusions: The distribution of streptomycin MICs in E. coli can be greatly influenced by the genes encoding resistance to streptomycin. The strA-strB genes are probably involved in conferring high-level resistance to streptomycin, whereas the opposite seems to be the case for the aadA gene cassettes. The low-level streptomycin resistance, caused by the presence of aadA gene cassettes in integrons, represents an obstacle in classifying E. coli as susceptible or resistant to streptomycin. Furthermore, the determination of an epidemiological cut-off value for surveillance purposes is also complicated by dissemination of integrons containing the aadA cassettes.

Keywords: epidemiological cut-off values , strA-strB , aadA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The awareness of antimicrobial resistance as an emerging problem worldwide is increasing. The new zoonosis directive 2003/99/EC1 forces EU member states to implement monitoring programmes that provide comparable data on the occurrence of antimicrobial resistance in zoonotic agents, and insofar as they present a threat to public health, other agents.

Several European countries, as well as Japan and the USA, have established monitoring programmes for antimicrobial resistance.2,3

Unfortunately, there is a lack of harmonization between the established national antimicrobial resistance monitoring programmes. Large differences are observed in the methodology used, the bacterial species and the antimicrobial agents tested.24

One of the major difficulties is the determination and harmonization of breakpoints needed for the classification of the bacterial isolates as susceptible or resistant. It has been shown that the harmonization of breakpoints used by laboratories in the Nordic countries can increase the percentage of concordance between the laboratories.4

The European harmonization of MIC breakpoints for antimicrobial susceptibility testing has recently been discussed.5 It was emphasized that epidemiological cut-off values are to be preferred for monitoring purposes, rather than using breakpoints designed for clinical purposes. Epidemiological breakpoints are determined on the basis of the distribution of MICs for each antimicrobial and bacterial species. The population that clearly departs from the normal susceptible population (‘wild-type’) is categorized as resistant (‘non-wild-type’). Further definitions can be found at the European Committee on Antimicrobial Susceptibility Testing (EUCAST) website (www.eucast.org). It is stated that ‘The agreement on the epidemiological cut-off values should not be difficult as the wild-type MIC distributions make these more or less self-evident’.5

However, it is not always easy to distinguish between the non-wild-type and the wild-type by looking at the MIC distributions of some antimicrobials and bacterial species. This applies to the MIC distributions of streptomycin for Escherichia coli.

In this study we have investigated the genetic background for resistance to streptomycin in a selection of E. coli strains that have been included in the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET). The isolates have previously been subjected to susceptibility testing and the MICs to streptomycin are known. From previous investigations we know that the strA-strB gene pair6 and the aadA gene cassette7 occur frequently among streptomycin-resistant E. coli of the normal intestinal flora of healthy domestic animals in Norway.8,9 The linked strA-strB gene pair mediates resistance to streptomycin by inactivation of streptomycin by two phosphotransferase enzymes (aminoglycoside-3''-phosphotransferase and aminoglycoside-6''-phosphotransferase).6 The strA-strB genes are widely disseminated among diverse Gram-negative bacteria and they have been detected in bacteria colonizing plants, animals, humans and farmed fish.8,10,11

The closely related aadA gene cassettes, located within multiresistance integrons, encode aminoglycoside adenylyltransferases inactivating streptomycin and spectinomycin.7 These gene cassettes are among the most prevalent gene cassettes in class 1 and class 2 integrons. The aadA gene cassettes have been detected in integrons in Gram-negative bacteria isolated from humans, animals, poultry, wild mammals and farmed fish.9,1215

The E. coli isolates investigated in this study have been screened for the presence of streptomycin resistance determinants and subsequent analysis of the MICs in relation to genetic content has been performed. The difficulties related to the setting of an epidemiological cut-off value of streptomycin for E. coli are discussed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The monitoring programmes of antimicrobial resistance in Norway, NORM (for humans) and NORM-VET (for food, feed and animals), have been running since the year 2000.

The NORM-VET monitoring programme investigated 944 E. coli isolates from meat and meat products of poultry (359), pork (295), cattle (190) and sheep (100) during the years 2000, 2001, 2002 and 2003.1619 Figure 1 shows the distribution of streptomycin MICs in all 944 strains. Strains with a streptomycin MIC of ≥16 mg/L (136 strains) were included in this study and investigated for the presence of the strA-strB genes and the aadA gene cassettes.



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Figure 1. Distribution (%) of MICs of streptomycin for all E. coli isolates from meat and meat products investigated in NORM-VET 2000–2003 (n=944). The vertical black line indicates a possible choice of an epidemiological cut-off value for streptomycin >8 mg/L.

 
Detection of the strA-strB genes

The PCR amplification of the linked strA-strB genes was carried out with the primers 5'-TAT-CTG-CGA-TTG-GAC-CCT-CTG-3' and 5'-CAT-TGC-TCA-TCA-TTT-GAT-CGG-CT-3'. The template was prepared by boiling a bacterial pellet in sterile distilled water and 5 µL of suspension was added to 45 µL of a mixture of PCR reagents containing 1xPCR buffer (Qiagen PCR Buffer; Qiagen GmbH, Hilden, Germany) with 1 U of Taq DNA polymerase (Qiagen), and 10 pmol of each primer and 200 µM of (each) dNTP. PCR was performed in a thermal cycler and the temperature profile was: DNA denaturation at 95°C for 60 s, then 30 cycles with 95°C for 60 s, followed by 60°C for 30 s and 72°C for 60 s. Positive and negative controls were used in each run. The size of a positive amplicon was 538 bp. An E. coli strain harbouring the plasmid RSF 1010 was used as a positive control,20 and E. coli DH5{alpha} as negative control. The size of the PCR products was evaluated after agarose gel electrophoresis.

Detection of the aadA gene cassettes

PCR was carried out with the primer pair 5'-GAG-AAC-ATA-GCG-TTG-CCT-TGG-3' and 5'-TCG-GCG-CGA-TTT-TGC-CGG-TTA-C-3' for detection of the aadA1a gene cassette and other closely related aadA gene cassettes (with the exception of aadA4 and aadA5). PCRs were carried out in 50 µL volumes and the composition of the reaction mixtures were as described for detection of the strA-strB genes. Boiled bacterial suspensions (5 µL) were used as template. The temperature profile was: DNA denaturation at 95°C for 60 s, then 30 cycles with 95°C for 60 s, followed by 48°C for 30 s and 72°C for 60 s. Positive and negative controls were used in each run. The size of a positive amplicon was 198 bp. An E. coli strain containing a class 1 integron, with the aadA1a cassette inserted, was used as a positive control9 (strain Se 131/GenBank accession no. AJ238350), and E. coli DH5{alpha} as negative control. The size of the PCR products was evaluated after agarose gel electrophoresis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The strA-strB genes and/or an aadA gene cassette were detected in 110 of the 136 (80.9%) strains investigated. The strA-strB genes were the most prevalent, detected in 90 strains. The aadA gene cassettes were detected in 29 strains. Nine of the strains harboured both the strA-strB genes and an aadA cassette. Strains harbouring the strA-strB genes (solely) had MICs ≥32 mg/L (MIC50=128 mg/L) (Figure 2). Strains possessing an aadA gene cassette (as the sole gene) had considerably lower MICs of ≤64 mg/L (MIC50=16 mg/L) (Figure 2). Strains harbouring both the strA-strB genes and an aadA gene cassette had higher MICs (MIC > 128 mg/L) than strains harbouring only the strA-strB genes, as shown in Figure 2. The isolates where neither the strA-strB genes nor an aadA gene cassette were detected, had an MIC50 of 16 mg/L. However, six isolates of this category had MICs>16 mg/L, as can be seen in Figure 2.



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Figure 2. Distribution (%) of MICs of streptomycin for E. coli isolates with an MIC ≥16 mg/L and harbouring the strA-strB genes (solely) (n=81) (black bars), an aadA1 gene cassette (n=20) (grey bars), both the strA-strB genes and an aadA gene cassette (n=9) (striped bars) and neither the strA-strB genes nor an aadA gene cassette (n=26) (dotted bars).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The high proportion of the strA-strB genes and/or the aadA genes detected in the E. coli strains investigated suggests that these genes play a major role in conferring resistance to streptomycin among E. coli of domestic animals in Norway.

The distribution of MICs between isolates harbouring only an aadA gene cassette (MIC50=16 mg/L) and isolates harbouring only the strA-strB genes (MIC50=128 mg/L) differed considerably. This suggests that the strA-strB genes are involved in conferring high-level resistance to streptomycin, whereas the opposite seems to be the case for the aadA gene cassettes. The 26 strains not possessing any of these two genes may harbour other genes mediating resistance to streptomycin, or the resistance may be conferred via chromosomal mutations that alter the ribosomal binding site of streptomycin.21 From an ongoing study in our laboratory we know that none of these 26 strains contains the integrase gene of class 1 or class 2 integrons (data not shown), indicating that resistance to streptomycin is not conferred by gene cassettes within integrons in these isolates.

Most monitoring programmes use a breakpoint value of >16 or >32 mg/L for classifying E. coli strains as resistant to streptomycin. In this study we have demonstrated that strains harbouring an aadA gene cassette can have MICs of 16 mg/L. Such strains may therefore be classified as susceptible to streptomycin even though they harbour a gene encoding an enzyme modifying streptomycin. In our study this misclassification concerned 13 isolates (9.9%) classified as susceptible, but harbouring an aadA gene cassette.

Other investigations have shown that streptomycin-susceptible E. coli and Salmonella enterica serotype Newport isolates can harbour aadA gene cassettes within integrons.22,23 We have also, in an ongoing study in our laboratory, detected the aadA gene cassettes in E. coli strains from meat with streptomycin MICs of 4 and 8 mg/L (data not shown). This indicates that strains possessing aadA gene cassettes can have MICs far below the breakpoint values normally used to classify a strain as susceptible to streptomycin. It has also been demonstrated that silent integron-borne aadA genes could be fully expressed when transferred to a new host.22

Gene cassettes in integrons can have a variable expression and this is caused by several factors; for example, whether or not the integron is located on a high copy number plasmid. Several versions of the integron promoter located in the 5' conserved segment of the integron exist causing differences in the strength of the promoter.24 The expression of a cassette in an integron containing more than one inserted cassette is also influenced by the position of the cassette. The expression weakens as the cassette is situated nearer the 3' conserved segment.21 All these factors may lead to considerable variations of MICs when gene cassettes in integrons are responsible for antimicrobial resistance.

This study shows that the distribution of streptomycin MICs among E. coli can be greatly influenced by the genes encoding resistance to streptomycin. The study further indicates that the strA-strB genes mediate substantially higher MICs than the aadA gene cassettes. Low-level resistance to streptomycin caused by the presence of the aadA gene cassettes represents an obstacle in classifying E. coli strains as susceptible or resistant to streptomycin. Furthermore, the determination of an epidemiological cut-off value for surveillance purposes is also complicated by dissemination of integrons containing the aadA cassettes.

The distributions of MICs for each antimicrobial and bacterial species alone may be influenced by genetic elements responsible for phenotypic expression of resistance. The surveillance programmes should ideally combine MIC distributions and genetic investigations for the classification of isolates as resistant or not. However, at the present stage there is still a lack of knowledge of possible resistance genes that may influence the MIC distributions for various bacterial species and antimicrobial agents. In most cases it is easy to distinguish between the wild-type and non-wild-type MIC distributions and thereby set an epidemiological cut-off value, as pointed out by Kahlmeter et al.5 Nevertheless, the awareness that certain genetic elements may affect the MIC distributions should be considered when deciding the epidemiological cut-off values. In our opinion, the epidemiological cut-off values should be chosen rather conservatively in case the distribution indicates that there might be genetic elements influencing the curve. However, the proportion of misclassified susceptible isolates would thereby increase. For surveillance purposes this is to be preferred, thus avoiding misclassification of resistant isolates.


    Acknowledgements
 
This work was supported by a grant from the Norwegian Research Council (grant No. 153080/I10).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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