Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii

Margarita M. Navia, Joaquím Ruiz, Anna Ribera, M. Teresa Jiménez de Anta and Jordi Vila*,

Departament de Microbiologia, IDIBAPS, Hospital Clínic, Facultat de Medicina, Universitat de Barcelona, Villarroel 170, 08036-Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of gyrA, gyrB and/or parC mutations, quinolone uptake, outer membrane protein profiles and epidemiological relationship were studied in 12 clinical isolates of Citrobacter freundii. No alterations were observed in the gyrB gene of any of the strains, or gyrA or parC of the four quinolone-susceptible strains (nalidixic acid MIC of 2–4 mg/L, and a ciprofloxacin MIC of 0.006–0.06 mg/L). The quinolone-resistant strains were classified into two groups: one group (group A) composed of strains resistant to nalidixic acid but not to ciprofloxacin and another (group B) including those resistant to both antibiotics with a mutation at codon 83 of the gyrA gene (Thr->Ile), but no alteration in either parC or gyrB genes. In group B, three of the four resistant isolates, with a nalidixic acid MIC > 1024 mg/L and ciprofloxacin MIC of 8–32 mg/L, showed concomitant mutations at codons 83 and 87 of the gyrA gene (Thr->Ile and Asp->Tyr, respectively) as well as a single mutation in codon 80 of the parC gene (Ser->Ile). The fourth isolate did not possess the mutation at codon 87 of gyrA. Two strains belong to the same clone and, although they had the same type of mutations in the gyrA and parC genes, showed different MICs of ciprofloxacin. This difference was related to an efflux pump mechanism. Mutations in the gyrA and parC genes play the main role in quinolone resistance development in Citrobacter freundii, although other factors such as overexpression of efflux pumps can play a complementary role and thus modulate the final quinolone MIC.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolones are a group of antimicrobial agents with a broad and potent activity spectrum which act by inhibiting the activity of type II topoisomerases (DNA gyrase and topoisomerase IV).1,2,3 Development of quinolone resistance can be due to amino acid substitutions in either or both of the two different subunits of the type II topoisomerases. These are both tetrameric enzymes composed of two subunits A and two subunits B, which in the case of DNA gyrase are encoded by the gyrA andgyrB genes, respectively. Quinolone resistance development owing to mutations in the gyrA gene has been well established.4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 In Escherichia coli, mutations are clustered in the quinolone resistance-determining region (QRDR) located between amino acids Ala-67 and Gln-106, the most frequent being at amino acid codon 8315 or at equivalent positions in other microorganisms including Citrobacter freundii.7,8,13,14,16,18The double mutation at amino acid codons Ser-83 and Asp-87 has been related to a high level of quinolone resistance in E. coli.6,9,17 Recently, the presence of mutations out of the QRDR (at amino acid 119 Ala->Glu) has been reported in quinolone-resistant Salmonella typhimurium.8 The mutation of amino acid codon Ala-119 to Ser has also been described in Acinetobacter baumannii,16 although no relationship has been established between this mutation and the presence of quinolone resistance, since it is present in both quinolone-resistant and quinolone-susceptible strains.

Mutations of amino acid codons 426 and 447 of the gyrB gene of E. coli are also responsible for the acquisition of quinolone resistance,10,19,20 although their relative importance differs in clinical and in-vitro E. coli quinolone-resistant strains. While quinolone-resistant strains show a similar in-vitro frequency of mutations in gyrA and gyrB genes,10 a clear predominance of gyrA gene mutations over those of gyrB gene has been shown in quinolone-resistant clinical isolates.12,17

There is a considerable sequence similarity between the genes that encode the A and B subunits of the DNA gyrase and those that encode them for the topoisomerase IV. Recently, it has been shown that topoisomerase IV is a quinolone target in E. coli3,21 and that changes at residues Ser-80 and Glu-84 of ParC (A subunit) may contribute to decreasing fluoroquinolone susceptibility.6,22,23,24Conversely, the role of ParE, (B subunit of topoisomerase IV) in the acquisition of quinolone resistance in clinical isolates of E. coli seems to be irrelevant,6,25 although mutations in in-vitro quinolone-resistant strains of E. coli have been described by Breines et al.26 in an analogous position to gyrB gene mutations responsible for quinolone resistance development.

Changes in quinolone accumulation by increased efflux or decreased uptake have also been linked to quinolone resistance acquisition. While the former is usually linked to some kind of pump which actively expels the drug, the latter has been associated with changes in the outer membrane proteins.

Alterations in the gyrA gene of a quinolone-resistant strain of C. freundii were shown for the first time indirectly with an experiment in which the supercoiling activity of the mutant's DNA gyrase was resistant to quinolone compounds.5 In 1988, Aoyama et al.27 studied norfloxacin uptake and membrane profiles in a clinically resistant strain and recently, mutations in gyrA and parC have been described in quinolone-resistant clinical strains.11 However, a comprehensive study of all possible resistance mechanisms in clinical isolates of C. freundii has not yet been reported.

In this work, we have determined the mechanisms of quinolone resistance in clinical isolates of C. freundii by means of sequencing the QRDR of the gyrA, gyrB and parC genes and determining quinolone uptake and outer membrane protein profiles.


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

A total of 12 clinical isolates of C. freundii were recovered from different biological samples, from either in-patients or out-patients, submitted to the Clinical Microbiology Laboratory at the Hospital Clinic of Barcelona, Spain.

Antimicrobial susceptibility testing

Susceptibility testing was performed by an agar dilution method in accordance with the guidelines established by the National Committee for Clinical Laboratory Standards.28 Approximately 104 cfu/spot of each isolate was inoculated with a multipoint replicator on to freshly prepared medium containing serial dilutions of ciprofloxacin (Bayer, Leverkusen, Germany), nalidixic acid (Prodesfarma, Barcelona, Spain) or chloramphenicol (Sigma, St Louis, MO, USA)

REP (repetitive extragenic palindromic) PCR

REP–PCR was carried out using the primer 5' GCG CCG ICA TGC GGC ATT 3' under the following conditions: 30 cycles of 1 min at 94°C, 1 min at 40°C, 1 min at 65°C and a final extension at 65°C for 16 min. The reaction was prepared using 5 µL of boiled bacterial suspension, 1 µL of 5 µM primer and a PCR bead (Pharmacia Biotech, Uppsala, Sweden) in a final volume of 25 µL. Five microlitres of the amplification product was separated in a 12.5% precast polyacrylamide gel using a GenePhor apparatus (Pharmacia Biotech) and silver-stained using the Pharmacia Biotech DNA silver staining kit.

Ciprofloxacin uptake

Ciprofloxacin uptake was determined as previously described by Asuquo & Piddock.29

Amplification and DNA sequencing of quinolone resistance determining region (QRDR) in gyrA and parC genes

The PCR amplification of the QRDR gyrA gene was carried out using the primers and following the conditions previously described by Vila et al.16 The parC QRDR was amplified using the sense primer described for E. coli23 and an antisense consensus primer designed by comparing several published parC gene sequences. The sequence of the antisense primer was 5'-CAT CGC CGC GAA CGA TTC GG-3'. The PCR conditions were the same as for gyrA amplification.16 To amplify the gyrB fragment, primers and conditions used were as described previously.16 The PCR reactions were performed using a DNA thermal cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA). Amplified DNA products were resolved by electrophoresis in agarose gels (2%, w/v) containing 0.5 mg/L of ethidium bromide. PCR products were recovered directly from the agarose gel and purified. DNA sequencing was performed with the TaqDyeDeoxyTerminator Cycle Sequencing kit (Applied Biosystems) and analysed in an automatic DNA sequencer (Applied Biosystems 377).

The EMBL accession numbers for the partial sequences are: gyrA, AF064797; parC, AF064798 and gyrB, AF071877.

Outer membrane protein profile analysis

Outer membrane proteins were prepared with N-lauroylsarcosine as previously described.30 Proteins were separated by electrophoresis using 10% urea-SDS-polyacrylamide gel and silver stained using the Pharmacia-Biotech protein silver staining kit.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
REP–PCR of the 12 C. freundii isolates showed eight different patterns (Figure 1). All the susceptible isolates and most of the nalidixic acid-resistant/ciprofloxacin-susceptible ones were different strains, with the exception of 16.0, 16.4 and 16.5 which showed the same pattern. The resistant strains can be grouped as pairs: isolates 1.2 and 1.25 were the same clone, as were isolates 1.44 and 1.38. These latter isolates came from the same patient.



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Figure 1 Silver stained 12.5% polyacrylamide gel electrophoresis of the REP–PCR of the 12 strains of C. freundii. Lane A, molecular weight marker; lane B, strain 1.38; lane C, strain 1.44; lane D, strain 1.2; lane E, strain 1.25; lane F, strain 14.1; lane G, strain 16.5; lane H, strain 16.4; lane I, strain 16.3; lane J, strain 16.0; lane K, strain 14.0; lane L, strain 8.0 and lane M, strain 18.0.

 
The PCR products obtained with the aforementioned primers for gyrA, gyrB and parC had the expected sizes, that is 343 bp, 447 bp and 240 bp, respectively. In the case of gyrA and parC, a fragment of the genes had been previously amplified.11 Our fragments had 100% homology with those described, although in both cases we sequenced a larger amplified fragment. As for gyrB, nucleotide identity with E. coli was 90.58% and between 90.42% and 54.38% with equivalent regions of other microorganisms while amino acid alignment showed 97.1% with E. coli and between 44.9% and 93.75% with the remainder (Figure 2).



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Figure 2 Comparison between the amino acid sequences of the sequences region of GyrB of C. freundii and the same region of four other microorganisms. The accession numbers of the sequence database for the GyrB proteins are: E. coli, D87842; S. typhimurium, Z68167; N. gonorrhoeae, M59981 and S. aureus, X71437.

 
The four quinolone-susceptible strains (nalidixic acid MIC 2–4 mg/L and ciprofloxacin MIC < 0.006–0.06 mg/L) all had the wild-type amino acid threonine at position 83 ofGyrA (Table I). The remaining eight strains were classified into two categories: (i) group A: four isolates resistant to nalidixic acid (MIC of 1024 mg/L) and susceptible to ciprofloxacin (MIC of 0.25–0.5 mg/L) and (ii) group B: four isolates resistant to both antibiotics (nalidixic acid MIC > 1024 mg/L, ciprofloxacin MIC 8–32 mg/L). Those included in group A showed only one substitution on GyrA (Thr-8->Ile), while three of the resistant isolates (group B) showed concomitant mutations that brought about the substitutions Thr-8->Ile and Asp-87->Tyr in GyrA plus an additional substitution Ser-80->Ile in ParC. The fourth isolate in group B only presented two mutations, one in gyrA (Thr-8->Ile) and one in parC (Ser-80->Ile). All the isolates had the expected Lys-447 and Asp-426 residues in GyrB.


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Table I. Relationship between REP–PCR group, MIC, gyrA and parC mutations for the twelve strains of Citrobacter freundii studied
 
The outer membrane protein profiles for the four isolates belonging to group D including the four resistant strains (group B), two susceptible strains (8 and 14) and two strains from group A (14.1 and 16.4) were investigated. No major differences were observed in the expression of OmpF, OmpC and OmpA (Figure 3 only shows the results for the four resistant strains). However, differences were observed in the amount of ciprofloxacin accumulated by the different strains tested (Table II). Strain 1.38 showed less accumulation than its pair strain 1.44 and the addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) produced an increase in the uptake of 51% for strain 1.38 and only of 8.7% for strain 1.44. The MIC of chloramphenicol for strain 1.38 was 128 mg/L and for strain 1.44 was 12 mg/L.



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Figure 3 Silver stained 10% urea-SDS-polyacrylamide gel electrophoresis of C. freundii outer membrane proteins. Lane 1, strain 1.2; lane 2., strain 1.25; lane 3, strain 1.38 and lane 4, strain 1.44.

 

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Table II. Ciprofloxacin accumulation in the presence and absence of CCCP in clinical isolates of C. freundii
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of gyrA, gyrB and parC mutations, differences in the ciprofloxacin uptake and OMP profiles of 12 clinical isolates of C. freundii have been analysed. In agreement with previously published results, all the nalidixic acid-resistant clinical isolates included in this study showed a mutation resulting in an amino acid substitution at the Thr-83 position (equivalent to Ser-83 of E. coli). Besides, three of them (ciprofloxacin MIC 8–32 mg/L) also showed a second substitution at the position equivalent to amino acid 87 of E. coli (Asp->Tyr). The four isolates that showed resistance to both quinolones employed, also had a mutation in the parC gene at the position equivalent to the residue Ser-80 of E. coli (Ser->Ile). We did not find the mutation at Glu-84 (Glu->Lys) described by Nishinoet al.11 No mutations were found in the analysed region of gyrB of any of the strains studied. These results suggest that in C. freundii, as in E. coli, the primary quinolone acquisition point is located in GyrA. Besides, a single substitution at position 83 of the GyrA is sufficient for developing a very high nalidixic acid resistance level (MIC >= 1024 mg/L), yet retaining susceptibility to ciprofloxacin (0.25–0.5 mg/L). This is clearly seen with isolates 16.0 and 16.4 or 16.5, which belong to the same REP–PCR type. The same has been described in E. coli15,17 where a change in Ser-83 was responsible for a nalidixic acid MIC of between 128 and 256 mg/L while ciprofloxacin remained with a MIC of 0.25 mg/L.

Even though the topoisomerase-mediated resistance (specifically mutations in gyrA and parC genes) can explain in general terms the MICs of the strains under study, differences within group B (resistant to both quinolones tested) need further explanation. C. freundii outer membrane proteins have been described previously.27 As in E. coli there are three major proteins, although their electrophoretic mobilities are different. A decrease in the expression of an outer membrane porin has been associated with a decrease in quinolone uptake.27 In our study, no major differences were observed in the expression of the isolates' OmpC, OmpF and OmpA profiles.

Differences did appear, however, in the amount of ciprofloxacin accumulated. Strains 1.44 and 1.38 belong to the same REP–PCR type, have the same gyrase and topoisomerase mutations but differ in their ciprofloxacin MIC. Such a difference could be attributed to strain 1.38 having a more important active efflux than 1.44, which is suggested by the former's increased ciprofloxacin uptake in the presence of CCCP. This suggestion is supported by the fact that the chloramphenicol MIC for strain 1.38 was higher than that for strain 1.44. This is probably due to the fact that the active efflux system pumping ciprofloxacin out of the cell also takes chloramphenicol.

In summary, in C. freundii, as in E. coli, high levels of quinolone resistance are due to mutations in gyrA and parC genes, with the former having the primary priority point mutation. In addition, an overexpressed active efflux pump responsible for increasing already high levels of resistance to ciprofloxacin was also observed in resistant strains.


    Acknowledgments
 
We thank the Servicios Científico Técnicos of the University of Barcelona for helping us with the DNA sequencing. This work was supported in part by the grant SAF 97/0091 from CICYT, Spain.


    Notes
 
* Corresponding author. Tel: +34-3-2275522; Fax: +34-3-2275454; E-mail: vila{at}medicina.ub.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 11 October 1998; returned 14 December 1998; revised 18 June 1999; accepted 27 July 1999