Molecular diversity of quinolone resistance in genetically related clinical isolates of Staphylococcus aureus and susceptibility to newer quinolones

G. Yagüe Guiraoa, M. C. Martínez Toldosa, B. Mora Perisa, M. A. Alonso Manzanaresb, M. N. Gutiérrez Zufiaurreb, J. A. Martínez Andrésa, J. L. Muñoz Bellidob, J. A. García-Rodríguezb and M. Segovia Hernándeza,*

a Departamento de Genética y Microbiología, Facultad de Medicina, Hospital General Universitario, Universidad de Murcia, Murcia; b Departamento de Microbiología, Hospital Universitario de Salamanca, Salamanca, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genes encoding topoisomerases (gyrA and grlA) and the norA promoter of 100 fluoroquinolone-susceptible and -resistant Staphylococcus aureus clinical isolates obtained in two geographically distant hospitals were analysed. The relationship between mutations found and the susceptibility to newer quinolones was determined. Thirty-nine strains were grouped in seven clones by pulsed-field gel electrophoresis (PFGE). The remaining 61 strains were classified as unrelated strains. In three clones, all strains showed the same grlA–gyrA–norA mutation profiles. Strains in the rest of the groups showed different mutation profiles, even though PFGE indicated that they possessed genetically similar populations. One cluster showed a high level of diversity; five different mutation profiles were detected in the six isolates belonging to this pattern. Two isolates had a Glu84 to Lys mutation in grlA and another isolate had this mutation combined with a Ser84 to Leu mutation in gyrA. Combination of a Ser80 to Phe mutation in grlA and a Ser84 to Leu in gyrA was found in the two other isolates. One of these also had a thymine to a guanine transversion at a position 89 nucleotides upstream of the norA start codon in the norA promoter. These results show that fluoroquinolone resistance in clinical S. aureus strains does not necessarily result from the spread of resistant clones. Fluoroquinolone resistance may develop independently in strains belonging to the same PFGE pattern by accumulation of different mutations over a quinolone-susceptible ancestor wild type or single grlA mutant.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolones are broad-spectrum, potent antimicrobial agents that have been widely used for more than a decade. Extensive clinical use of these agents has led to an increase of resistant organisms, and reports of a high prevalence of fluoroquinolone resistance among Staphylococcus aureus isolates have appeared, particularly in methicillin-resistant S. aureus.1,2

Among the various mechanisms of quinolone resistance, mutations in grlA, which encodes the GrlA subunit of DNA topoisomerase IV, appear to play a central role in conferring quinolone resistance in S. aureus.3 Recently, grlB mutations have been reported in S. aureus, but the effect of these changes is unclear.4 In addition, mutations in gyrA and gyrB, which encode subunits of DNA gyrase, seem to play a complementary role in increasing resistance to fluoroquinolones in this species.5,6 Current studies have demonstrated that in S. aureus and other Gram-positive bacteria such as Streptococcus pneumoniae, topoisomerase IV is the primary target of fluoroquinolones.7,8 In addition, another mechanism of low-level resistance is the active efflux mediated by increased transcription of the norA gene,9 the gene encoding a multidrug efflux protein capable of transporting fluoroquinolones. Topoisomerase- and NorA-mediated resistance mechanisms can occur alone or in combination.10

Little is known about how changes in the genes encoding topoisomerases and NorA appear and spread in staphylococci. In some reports, the high prevalence observed within particular institutions has proved to be related to cross-infection of patients by a limited number of resistant clones.11 However, in a recent study a single clone of Enterococcus faecalis was found to have distinct mutations in the gyrase genes.12 In the present work, we have analysed the genes encoding topoisomerases and the norA promoter in fluoroquinolone-susceptible and -resistant S. aureus clinical isolates belonging to seven clones, isolated in two geographically distant hospitals; the relationship between the mutations was found and the susceptibility to newer fluoroquinolones determined.


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

One hundred S. aureus clinical isolates from in-patients [43 methicillin resistant (MRSA) and 57 methicillin susceptible (MSSA)] collected successively during a 1 year period from two general hospitals in Spain—Hospital General de Murcia (in the southeast of Spain, 55 strains) and Hospital Universitario de Salamanca (in the mid-west of Spain, 45 strains)—were characterized by genotyping and antimicrobial susceptibility. Isolates were selected on the basis of the MICs of ciprofloxacin for these strains. We selected 33 clearly susceptible isolates (MICs of c. 0.1–0.2 mg/L) and 67 resistant isolates, for some of which the MICs were around the breakpoint (MICs of c. 1–2 mg/L) and some highly resistant isolates (MICs of >4 mg/L).

Analysis of total DNA by pulsed-field gel electrophoresis (PFGE)

Contour-clamped homogeneous electric field electrophoresis of SmaI restriction endonuclease digest of genomic DNA was performed with a CHEF-DR II system (BioRad Laboratories, Hercules, CA, USA) as described previously.13 Criteria for comparing the patterns obtained were as given in Tenover et al.14

Antibiotic susceptibility testing

Ciprofloxacin (Bayer, Spain), sparfloxacin (Rhône-Poulenc Santé, Spain), levofloxacin (Hoechst Marion Roussel, Spain) and trovafloxacin (Pfizer, Spain) were provided as standard powder by their respective manufacturers. The in vitro activity of these quinolones was studied by the standard agar dilution method, according to NCCLS guidelines.15

Detection of mutations

PCR was used to amplify DNA from the quinolone- resistance determining region (QRDR) of the gyrase (gyrA) and topoisomerase IV (grlA) genes and norA promoter gene. The PCR products were sequenced by standard methods. Chromosomal DNA was prepared by the method of Sambrook et al.16 Amplification of the QRDR of gyrA, grlA and the norA promoter was achieved using PCR with primers based on published sequences for these genes.17 A 124 bp fragment of the grlA gene, a 254 bp fragment of gyrA and a 257 bp DNA fragment of the norA promoter were obtained from each strain by PCR and sequenced according to previously described methods18 in order to identify specific mutations. Wild-type sequences and mutations were identified on the basis of comparison with the published sequences of grlA,7 gyrA 19 and the norA promoter.20

Northern blotting

To perform Northern blotting, total cellular RNA was prepared from cells of S. aureus by the guanidinium thiocyanate–phenol–chloroform procedure21 modified for S. aureus by use of lysostaphin (500 mg/L) to digest the cell wall and SDS (10%) and proteinase K (10 mg/mL) to lyse protoplasts. Equivalent amounts of RNA (20 µg) from each strain were applied and separated in a formaldehyde-containing agarose gel. The RNA was transferred on to a nylon membrane (Amersham, UK) for Northern blot analysis. The norA DNA probe was prepared by PCR and was labelled with [{alpha}-32P]dGTP by the random prime method (Rediprime DNA, Amersham). Hybridization and washes were carried out according to Sambrook's protocols.16


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PFGE analysis

Sixty-one of the isolates were shown by PFGE to have 61 different RFLP profiles and were considered as not clonally related. The remaining 39 strains were clustered in seven different PFGE patterns (Table IGo). Isolates belonging to the same pattern showed identical PFGE profile and were considered to be clonally related. In four out of these seven clusters (I, 10 strains; II, 13 strains; III, 6 strains and IV, 2 strains) we found isolates from the two different geographical locations (Table IGo). The other three clusters hold only Murcia isolates.


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Table I. Topoisomerase QRDR mutations and norA promotor mutations of 39 isolates of S. aureus belonging to SmaI PFGE groups
 
Characterization of mutations in topoisomerase genes and the norA promoter

The mutations identified are summarized in Table IGo. In three clones (II, V and VII) all strains showed identical grlAgyrAnorA mutation profiles. S. aureus clinical isolates assigned to PFGE patterns I, III, IV and VI, had distinct mutations in the topoisomerase genes and norA promoter. Wild-type isolates, grlA mutants, grlA–gyrA double mutants and norA promoter mutants were found in isolates belonging to the same PFGE pattern.

PFGE pattern III showed the highest level of diversity. Five different mutations were detected in the six isolates belonging to this pattern. No silent mutations were found. Three isolates came from Murcia and the remaining two from Salamanca. The S29 isolate had a thymine to guanine transversion at a position 89 nucleotides upstream of the norA start codon in the norA promoter. Northern hybridization showed a much stronger signal for this isolate in comparison with isolates with the wild-type norA promoter sequence. In this PFGE cluster we found methicillinsusceptible and -resistant strains. Strains contained in each of the remaining clusters had the same methicillin susceptibility.

Relationship between topoisomerase and norA promoter mutations, and susceptibilities to quinolones

Table IIGo shows the susceptibilities of the 39 isolates that had comprehensive fluoroquinolone susceptibility tested. When all the genes studied were considered, seven combinations of mutations were found (Table IIGo). In the three strains harbouring a single grlA mutation (Ser80 to Phe or Glu84 to Lys), MICs of fluoroquinolones increased moderately with respect to wild-type strains, resulting in low-level resistance to ciprofloxacin. These strains remained sensitive to newer fluoroquinolones.


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Table II. Relationship between mutations found in grlA, gyrA and the norA promoter and susceptibility to fluoroquinolones in 39 clinical isolates of S. aureus
 
There was no isolate that possessed gyrA mutations in the absence of a grlA mutation. The only GyrA modification found was the common Ser84 to Lys change that is always detected in combination with grlA mutations (Ser80 to Phe, 18 strains; Ser80 to Tyr, three strains; Glu84 to Lys, one strain). In these grlAgyrA double mutants, MICs of fluoroquinolones tested increased significantly.

The ciprofloxacin MIC in isolate S29 (norA promoter mutation) was high in comparison with that for isolate S28. Both isolates belonged to the same PFGE group (III) and had the same combination of mutations in topoisomerase genes.We also observed increased levels of norA mRNA in S29.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous work22 has shown that grlA or gyrA mutations responsible for quinolone resistance are usually restricted to only one or two point mutations at limited positions. Single grlA mutants have only moderately increased fluoroquinolone MICs, and remain sensitive to newer fluoroquinolones. MICs of fluoroquinolones are significantly increased in grlA–gyrA double mutants, which remain resistant to ciprofloxacin and, in most instances, to newer fluoroquinolones tested.

NorA-mediated quinolone efflux has been studied extensively in S. aureus. Previous studies have shown that norA overexpression can lead to fluoroquinolone resistance, both in the presence and in the absence of topoisomerase alterations.20,23

The high level of fluoroquinolone resistance found in isolate S29 to the less recently developed fluoroquinolones is associated with increased levels of norA mRNA. Uptake, accumulation of fluoroquinolones and ciprofloxacin MIC in the presence of reserpine have been determined previously in this strain.17 Ciprofloxacin uptake in the mutant promoter norA strain (S29) was significatively lower than in similar topoisomerase gene mutant strains and this uptake increased in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). S29, with its double gyrAgrlA mutation and the change at position –89 in the norA promoter, is more resistant to levofloxacin and sparfloxacin than the equivalent gyrAgrlA double mutant, the norA promoter isolate (S28). Overexpression of norA has been related to mutations 89 bp upstream of the putative ATG start codon,9,20 but may also happen independently of this mutation,23 suggesting that norA regulation can be located elsewhere in the chromosome. Recent studies suggest that this mutation might be necessary for constitutively increased norA expression, though not when overproduction is inducible.10

Trovafloxacin was the newer fluoroquinolone that showed the best activity against S29. It was eight- to 16-fold more active that the other newer fluoroquinolones against this isolate. Thus, norA overexpression, when it occurs in isolates with mutations in gyrA and grlA, can increase significantly the MICs of some newer fluoroquinolones, and this increase can be varied among newer fluoroquinolones. While some newer fluoroquinolones such as levofloxacin had MICs eight-fold higher, and were categorized as resistant (MIC >= 8 mg/L) according to NCCLS breakpoints, other newer fluoroquinolones such as trovafloxacin show a much lower increase of MICs and remain active even against this isolate.

Fitzgibbon et al.24 recently demonstrated that, in the absence of norA promoter mutations, high-level resistance to trovafloxacin required three QRDR mutations (grlA-80 and -84, and gyrA-84). Our results suggest that the combination of such mutations is needed to produce resistance to trovafloxacin, since we found that neither the most common double gyrA–grlA mutation nor these mutations combined with the change in the norA promoter was sufficient to produce resistance to trovafloxacin.

Kaatz & Seo10 recently showed that S. aureus strains obtained in vitro from the same parent strain can harbour most of the mechanisms of resistance known, individually or in combination, and that mutations resulting in topoisomerase- and NorA-mediated fluoroquinolone resistance can arise, alone or in combination, in a single step. In contrast, Ferrero et al.,25 analysing a stepwise-selected ciprofloxacin-resistant S. aureus mutant strain, demonstrated that mutations in gyrA or norA appeared only after alteration of GrlA.

High prevalence of fluoroquinolone resistance in S. aureus clinical strains observed within particular institutions has usually been considered to derive from the spread of a limited number of resistant clones.11 Nevertheless, some authors12 have demonstrated in other species, such as Enterococcus faecalis, that apparently genetically related clinical isolates can show different mutation patterns in the gyrase genes. Our results indicate that this also occurs in S. aureus. In PFGE group III, isolates from one hospital had the same grlA mutation, and this mutation was different from the mutations observed in isolates from the other hospital. This suggests that these isolates were derived from a wild-type common ancestor which evolved in a different way in each location. Hence, resistance to fluoroquinolones in clinical isolates of S. aureus can develop not only from the spread of resistant clones,2 but can also emerge by independent genetic events in previously quinolone-susceptible clones.


    Acknowledgments
 
This work was supported by a grant from the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad (grant FIS-96/1802).


    Notes
 
* Correspondence address. Departamento de Microbiología, Facultad de Medicina, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain. Tel: +34-968-360953; Fax: +34-968-360950. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 26 April 2000; returned 25 July 2000; revised 14 September 2000; accepted 12 October 2000