Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery

Francis Martineaua,b, François J. Picarda, Louis Greniera, Paul H. Roya,c, Marc Ouellettea,b, Michel G. Bergerona,b,* and the ESPRIT Trial,{dagger}

a Centre de Recherche en Infectiologie de l'Université Laval, Sainte-Foy, Québec, Canada G1V 4G2 b Division de Microbiologie, Faculté de Médecine c Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2''), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The number of patients undergoing coronary artery bypass graft (CABG) surgery worldwide has increased over the last two decades to >800000 patients annually.1 In Canada, >50000 CAGB procedures were performed between 1992 and 1995.2 Infectious complications are a major source of post-operative morbidity and mortality. The most common pathogens involved in these infections are Staphylococcus aureus, coagulase-negative staphylococci (CNS) and Gram-negative bacilli.3 Infections range from superficial involvement of the wound to deeper soft tissue infections such as sternal osteomyelitis and mediastinitis, requiring surgical drainage and debridement. Cardiac surgical patients are also at risk of developing infections unrelated to the surgical site, such as pneumonia, urinary tract infections and bacteraemia.

Empirical antibiotic regimens are needed to stabilize patients suffering from staphylococcal infections. The diagnosis of these infections depends largely on time-consuming (>=36 h) classical microbiological and biochemical methods for species identification and susceptibility testing.4 The prevalence of staphylococcal resistance to antimicrobial agents is alarmingly high and is still increasing. Consequently, the development of rapid and simple diagnostic tests that could identify staphylococcal species and determine their resistance profile would be an important improvement in the diagnosis of these infections and would help clinicians to treat them more efficiently.4

The usefulness of polymerase chain reaction (PCR)-based assays for the rapid detection of methicillin-resistant S. aureus is well established.58 Some PCR assays for detecting staphylococcal genes encoding aminoglycoside-modifying enzymes or erythromycin ribosomal methylases have also been developed.9,10 The aims of this study were: (i) to develop rapid multiplex PCR assays allowing simultaneous species-specific identification of S. aureus and Staphylococcus epidermidis and detection of seven associated clinically relevant antibiotic resistance genes; (ii) to compare those PCR assays with standard microbiological methods for staphylococcal species identification and susceptibility testing; and (iii) to study the incidence of these resistance genes in staphylococci associated with infections after cardiac surgery.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient population and bacterial strains

In this study, 82 S. aureus and 114 CNS (including 84 S. epidermidis) were isolated from 149 patients who developed severe infections after cardiac surgery. These patients were part of a population of 3027 adult patients undergoing elective CABG and/or valve surgery (the Elective Sternotomy Prophylaxis of Infection with Teicoplanin (ESPRIT) trial) who were followed for the incidence of infection after cardiac surgery. Thirteen Canadian cardiac surgical centres affiliated with universities participated in this study. A total of 196 staphylococcal strains from different infected sites (sternal and leg wound, blood, urine and sputum) were sent to the Infectious Diseases Research Center (IDRC) of Laval University (Sainte-Foy, Québec, Canada). The identification of all staphylococcal strains was reconfirmed at the IDRC by using the MicroScan Autoscan-4 system (Dade Diagnostics, Mississauga, Ontario, Canada).11

Antimicrobial susceptibility testing

Disc diffusion tests were performed for each of the 196 isolates identified as S. aureus, S. epidermidis, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus simulans or Staphylococcus warneri using the method recommended by the NCCLS.12 MICs of gentamicin and oxacillin were determined by the broth microdilution methodology recommended by the NCCLS.13 The chromogenic cephalosporin nitrocefin test was performed as recommended by the manufacturer (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA).

Multiplex PCR

The S. aureus- and S. epidermidis-specific PCR assays used in this study have been described previously.11,14 The PCR primers for the antibiotic resistance genes blaZ, mecA, aac(6')-aph(2''), ermA, ermB, ermC and msrA were chosen from database sequences (Table IGo). We have developed seven different multiplex PCR assays all including both species-specific primer pairs and a primer pair for the specific detection of one antibiotic resistance gene. Multiplex PCR amplifications were performed from a standardized bacterial suspension as described previously.11 An internal control was integrated into every PCR to verify the efficiency of the amplification and to ensure that significant PCR inhibition was absent.15


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Table I. Antibiotic resistance gene-specific and species-specific primers
 
In vitro selection of cells with increased resistance to gentamicin

Cells with increased gentamicin resistance were selected from strains that carried a gentamicin resistance gene but were sensitive to this antibiotic (based on disc diffusion and MIC determination) by subculturing them sequentially on to media containing increasing concentration gradients of gentamicin.16 Plates with a low (0–1 mg/L), intermediate (0–5 mg/L) or high (0–10 mg/L) gradient of concentrations were prepared. For subculturing, colonies in the area of highest antibiotic concentration were picked and streaked on to medium containing the same or a greater gradient of antibiotic concentrations. For these experiments, S. aureus strain ATCC 25923, which is susceptible to gentamicin and does not carry any of the antibiotic resistance genes tested in this study, was always used in parallel as a control.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Correlation between PCR and susceptibility testing

Results from the seven multiplex PCR assays correlated very well with those from conventional susceptibility testing (disc diffusion and MIC determination) and identification methods (the MicroScan system). There was perfect correlation between the results of PCR and classical biochemical methods for the identification at the species level of all S. aureus and S. epidermidis strains used in this study. There was no discordance between conventional susceptibility testing and PCR for the 82 S. aureus strains (Table IIGo). For CNS, there were four discordances for gentamicin resistance and two for oxacillin resistance (Table III). Four S. epidermidis strains carrying the resistance gene aac(6')-aph(2'') based on PCR were susceptible to gentamicin according to the disc diffusion method and MIC determinations (MICs of 4 mg/L for all). Although the aac(6')-aph(2'') gene was present, gentamicin resistance was not detected by classical susceptibility testing methods (Table III). Three S. epidermidis and one S. hominis were all mecA+ but susceptible to oxacillin based on disc diffusion. However, these strains were all resistant to oxacillin based on MIC determination. It is clear that the disc diffusion method gave erroneous results for these four CNS strains. Also, two CNS strains (one S. epidermidis and one S. warneri) were resistant to oxacillin but did not carry mecA (Table III). These two strains were further characterized by nitrocefin testing. A positive reaction with the nitrocefin test and the presence of blaZ for both strains confirmed that they were ß-lactamase producers.


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Table II. Correlation between antibiotic susceptibility testing and the presence of clinically relevant antibiotic resistance genes in 82 strains of S. aureus
 
In vitro selection of cells with increased resistance to gentamicin

The four S. epidermidis strains positive for aac(6')-aph(2'') but susceptible to gentamicin (MIC 4 mg/L) were subcultured on plates containing increasing gradients of gentamicin. This allowed the selection of cells with MICs of gentamicin ranging from 16 to 64 mg/L. We confirmed by PCR that aac(6')-aph(2'') was still present in these four strains after the selection process. It was not possible to select cells with increased MICs of gentamicin with the control S. aureus strain ATCC 29213 (its MIC remained at 0.5 mg/L), showing that the gentamicin resistance gene must be present in order to allow selection of cells with increased resistance.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This paper describes multiplex PCR assays that can be used to detect clinically relevant antibiotic resistance genes frequently encountered in staphylococci. These assays were applied to staphylococcal strains associated with multiple site infections after cardiac surgery. We have compared the performance of these PCR assays with those of classical methods of susceptibility testing and species identification. Overall, we found a correlation between phenotypic and genotypic methods of 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin G resistance and 100% for S. aureus and S. epidermidis species identification. Although S. epidermidis is the CNS species that causes most infections after cardiac surgery, 30 of the 114 isolates in this study were other species and would not be identified by PCR unless primers specific for these species were used.

Erythromycin resistance in staphylococci is predominantly mediated by erythromycin resistance methylase encoded by erm genes.17 In human infections caused by staphylococci, ermA and ermC are the most common methylase genes.18 Staphylococcal strains resistant to macrolides and type B streptogramins also frequently harbour msrA, which encodes an ATP-dependent efflux pump.10,19 In the present study, the incidence of ermA was 12.2% for S. aureus and 4.4% for CNS. A similar incidence for ermA has been reported for CNS isolated from various sites and specimens.19 Interestingly, all erythromycin-resistant S. aureus from the present study carried ermA. Others have also observed high incidences (82–94%) for ermA in erythromycin-resistant S. aureus isolated from blood.18,20 In contrast, we found no staphylococci carrying ermB. This is in agreement with the study of Eady et al.,19 which showed that ermB was found in animal CNS isolates but not in CNS of human origin. For ermC, we found an incidence of 1.2% for S. aureus and 36.8% for S. epidermidis. A similar incidence (51%) of ermC in CNS of various origins has been reported previously.19 Erythromycin-resistant isolates carrying ermC from the present study represented 10% of S. aureus and 85.7% of CNS. Others have found similar incidences in erythromycin-resistant S. aureus blood isolates.18,20 Westh et al.21 also reported a high incidence (76%) of erythromycin-resistant CNS strains carrying ermC. Finally, we found that none of the S. aureus strains and 5.3% of CNS harboured the msrA gene. A similar incidence for msrA in CNS (4.1%) of various origins has been reported in the UK.19

aac(6')-aph(2'') encodes the aminoglycoside-modifying enzyme AAC(6')-APH(2''), which confers resistance to gentamicin and concomitant resistance to tobramycin and kanamycin.22 aac(6')-aph(2'') is the most frequently encountered aminoglycoside resistance gene in staphylococci.9,23 In this study, we looked at the correlation between gentamicin resistance and the presence of aac(6')-aph(2''). We found aac(6')-aph(2'') in 6.1% of S. aureus isolates and 36.8% of CNS isolates. Importantly, all staphylococcal strains resistant to gentamicin in the present study carried the aac(6')-aph(2'') gene. Other investigators have found an excellent correlation between the presence of aac(6')-aph(2'') and resistance to gentamicin based on MIC determination or disc diffusion.9 A similar incidence for gentamicin resistance has been reported in the USA and Canada.24 This suggests that staphylococci associated with infections after cardiac surgery do not differ significantly from strains from other infections in the distribution of the resistance gene aac(6')-aph(2''). Four S. epidermidis strains that carried the aac(6')-aph(2'') were susceptible to gentamicin based on disc diffusion and MIC determination. However, these strains were borderline (MIC 4 mg/L) (typical MICs of gentamicin for susceptible S. epidermidis are in the range 0.015–0.125 mg/L). The much higher gentamicin MIC of these strains is probably attributable to the expression of aac(6')-aph(2'').

The mecA gene is a 2.4 kb chromosomal determinant encoding a penicillin-binding protein with a reduced affinity for all ß-lactam antibiotics.25 Several studies deal with the detection by PCR of the mecA gene only,6,8 or combined in multiplex with S. aureus-specific amplification.5,7 Detection of mecA by PCR is now considered the gold standard method, mainly because phenotypic methods using oxacillin may be difficult to interpret and some isolates do not express their mecA gene unless selective pressure via antibiotic treatment is applied.26 Overall, we found in this study that 6.1% of S. aureus and 53.5% of S. epidermidis strains carried mecA. These incidences for oxacillin resistance are similar to those reported previously in Canada and the USA.24 Two ß-lactamase-producing strains (carrying blaZ) of CNS were borderline oxacillin-resistant based on MIC determination but did not harbour the mecA gene. Methicillin resistance in these strains may be associated with ß-lactamase hyperproduction or with other resistance mechanisms.26

ß-Lactamase production in staphylococci is encoded by blaZ.27 We found that 62.2% of S. aureus and 79.8% of S. epidermidis isolates carried blaZ, with no discrepant result with the disc diffusion method. The incidence of penicillin resistance found in the staphylococcal isolates from the present study is similar to that from the SENTRY Antimicrobial Surveillance Program in Canada and the USA.24 No penicillin G-resistant strains had the resistance genotype mecA+/blaZ. None of the strains carrying blaZ was susceptible to penicillin G, suggesting that blaZ was efficiently expressed in all blaZ+ strains tested in this study.

We have confirmed the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections. Our results also suggest that staphylococci associated with post-operative cardiac surgery infections do not have a distinctive incidence or distribution of the clinically relevant antibiotic resistance genes studied. Our findings with the selection of cells with higher gentamicin MICs from strains defined as susceptible by classical susceptibility testing methods suggest that a susceptible strain harbouring an antibiotic resistance gene should be regarded as potentially resistant to that antibiotic. This particular situation may be explained by one or more mutations in the regulatory region or in the structural gene attributable to a lack of selective pressure for gentamicin resistance. Others have made similar observations for oxacillin resistance in S. aureus.28

Although classical susceptibility testing methods are relatively simple, they require bacterial isolation, so the results are not available for >=2 days, long after treatment has been started.4 Classical methods for susceptibility testing have additional shortcomings: (i) since different bacterial species differ in their susceptibility to a given antibiotic, breakpoints of different values must be tested; (ii) there is no international agreement for the interpretation of breakpoints in antibiotic susceptibility tests; and (iii) susceptibility tests may be highly dependent on experimental conditions. We believe that one direct impact of PCR assays such as those described in this study is their potential to allow more rapid establishment of effective antibiotic therapy. These multiplex PCR assays could be adapted for direct detection from positive blood cultures or from normally sterile clinical specimens (e.g. blood or urine), thereby allowing diagnosis to be made much more quickly than by conventional culture methods. These assays could also be optimized for direct detection from nasal or wound swabs, but would be of limited value for specimens in which a mixture of S. aureus and CNS is commonly found. Overall, these assays should contribute to a reduction of empirical treatment with broad-spectrum antibiotics, which are associated with high cost and toxicity.4 The consequent reduction of antibiotic use should help to reduce the emergence of resistance.


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Table III. Correlation between antibiotic susceptibility testing and the presence of clinically relevant antibiotic resistance genes in 11 4 strains of CNS
 

    Acknowledgments
 
We thank members of the ESPRIT trial for providing all staphylococcal strains. We also thank Ann Huletsky and Christian Ménard for helpful suggestions. F. Martineau has a scholarship from Fonds de Recherche en Santé du Québec. M. Ouellette is a Medical Research Council Scientist and a Burroughs Wellcome Fund New Investigator in molecular parasitology. This research project was supported by grant PA-15586 from the Medical Research Council of Canada and by Infectio Diagnostic Inc. (IDI), Sainte-Foy, Québec, Canada. The ESPRIT trial involved the following Canadian investigators: P. Auger (Cardiology Institute of Montréal, Montréal, Québec), I. Luch-singer and S. J. Landis (Hamilton General Hospital, Hamilton, Ontario), F. Aoki (Health Science Centre, Winnipeg, Manitoba), R. Saginur (Heart Institute of Ottawa, Ottawa, Ontario), M. G. Bergeron (Laval Hospital, Sainte-Foy, Québec), D. Portnoy (Montréal General Hospital, Montréal, Québec), H. Robson (Royal Victoria Hospital, Montréal, Québec), Y. Girouard (Sacré-Coeur Hospital, Montréal, Québec), I. Salit (Toronto General Hospital, Toronto, Ontario), S. Shafran (University of Alberta, Edmonton, Alberta), A. Chow (Vancouver General Hospital, Vancouver, British Columbia), W. Schlech (Victoria General Hospital, Halifax, Nova Scotia) and M. Myers (Victoria Hospital, London, Ontario).


    Notes
 
* Correspondence address. Centre de Recherche en Infectiologie, CHUQ (Pavillon CHUL), 2705 Boulevard Laurier, Sainte-Foy, Québec, Canada, G1V 4G2. Tel: +1-418-654-2705; Fax: +1-418-654-2715; E-mail: michel.g.bergeron{at}crchul.ulaval.ca Back

{dagger} The Canadian investigators involved in the ESPRIT trial are listed in the Acknowledgements. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 4 November 1999; returned 6 February 2000; revised 26 April 2000; accepted 5 June 2000