Frequencies and mechanisms of resistance to moxifloxacin in nosocomial isolates of Acinetobacter baumannii

Richard P. Spence and Kevin J. Towner*

Molecular Diagnostics and Typing Unit, Department of Microbiology, University Hospital, Queens Medical Centre, Nottingham NG7 2UH, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives. To compare the in vitro activity of moxifloxacin and ciprofloxacin against 226 nosocomial isolates of Acinetobacter baumannii from 44 hospitals in the UK.

Methods: MICs of ciprofloxacin and moxifloxacin were determined by Etest. PCR analysis was used to detect chromosomal mutations in the gyrA and parC genes. Isolates resistant to ciprofloxacin and susceptible to moxifloxacin were examined for the ability to generate spontaneous moxifloxacin-resistant isolates.

Results: Of 226 isolates, 49.1% were resistant to ciprofloxacin and 39.4% were moxifloxacin-resistant according to BSAC criteria. Approximately 20% of isolates resistant to ciprofloxacin remained susceptible to moxifloxacin. A GyrA mutation at Ser-83 was found in all ciprofloxacin-resistant isolates. Single mutations in both the gyrA and parC genes at codons Ser-83 and Ser-80, respectively, were found in ciprofloxacin- and moxifloxacin-resistant isolates. Isolates that were ciprofloxacin-resistant but moxifloxacin-susceptible generated spontaneous moxifloxacin-resistant mutants when grown on medium containing up to 8x their initial MIC. However, these mutants were not stable and none displayed high-level moxifloxacin resistance.

Conclusions: Moxifloxacin retained in vitro activity against some ciprofloxacin-resistant clinical A. baumannii isolates. Mutations in both gyrA and parC were necessary for resistance to moxifloxacin in most isolates of A. baumannii.

Keywords: Acinetobacter, fluoroquinolones, gyrA, parC, resistant


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Outbreaks of infection caused by Acinetobacter baumannii have become an increasing problem over recent years, especially in the hospital setting.1 Many strains possess multiple antibiotic-resistance mechanisms, spread readily between patients and are capable of long-term survival on dry surfaces.1 Fluoroquinolones have been used effectively to treat A. baumannii infections, but many isolates are now resistant to most of these agents.2 Resistance normally involves chromosomal mutations in the quinolone resistance determining regions (QRDRs) of either one or both of the DNA gyrase or topoisomerase IV genes that represent the primary and secondary intracellular targets for this class of antibiotic,3,4 but a recent study has suggested that a non-specific efflux pump mechanism could also contribute to quinolone resistance in A. baumannii.5

Newer fluoroquinolones, such as moxifloxacin, may have increased activity against A. baumannii in vitro in comparison with older agents such as ciprofloxacin.6,7 The present study compared the in vitro activity of these two compounds against A. baumannii isolates obtained from hospitals throughout the UK. The mechanism(s) of resistance to moxifloxacin were analysed and compared with those reported previously,3,4,6,7 and the frequency at which moxifloxacin-susceptible A. baumannii isolates yielded resistant mutants was assessed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and susceptibility testing

Two hundred and twenty-six clinical isolates of A. baumannii sensu stricto were obtained from 44 hospitals throughout the UK and were identified to the genomic species level by tDNA and AFLP fingerprinting.8 Isolates included in the study were a combination of outbreak-related and sporadic isolates belonging to the 35 RAPD genotypes and 15 outliers identified previously.8 Susceptibilities against ciprofloxacin and moxifloxacin were initially established using the British Society for Antimicrobial Chemotherapy (BSAC) standardized disc susceptibility testing method.9 MICs of ciprofloxacin-resistant and moxifloxacin-resistant isolates were determined with Etest strips (AB Biodisk, Solna, Sweden). Breakpoints used were those recommended for Acinetobacter spp. and Enterobacteriaceae by the BSAC.9,10 Escherichia coli (strain ATCC 25922) was used as the control strain for disc susceptibility testing and MICs as recommended by the BSAC.9

DNA extraction, amplification and digestion of the QRDR of gyrA and parC genes

Isolates resistant to ciprofloxacin and/or moxifloxacin were examined for mutations in the gyrA and parC genes at codons Ser-83 and Ser-80, respectively, as described previously.3,4

Selection of spontaneous single-step mutants

A. baumannii isolates resistant to ciprofloxacin but moxifloxacin-susceptible were examined for the generation of spontaneous single-step mutations to moxifloxacin resistance. Ten-fold dilutions of an overnight broth culture of each isolate (107–108 cfu/mL) were spread (100 µL portions) on to unsupplemented IsoSensitest agar plates (90 mm diameter), and IsoSensitest agar plates containing moxifloxacin at 1, 2, 4, 8 and 16x MIC for each respective isolate. Plates were incubated aerobically at 37°C for 48 h. The mutation frequency was calculated as the number of resistant colonies divided by the total viable count. Resistant colonies obtained at the highest antibiotic concentration for each respective isolate were screened for high-level moxifloxacin resistance by re-streaking on to IsoSensitest agar plates containing moxifloxacin 8 mg/L. MICs for colonies growing on moxifloxacin 8 mg/L were determined with Etest strips. Resistant colonies were also examined for mutations in the gyrA and parC genes.3,4 The stability of resistance was investigated by subculturing a single resistant colony on to IsoSensitest agar lacking moxifloxacin, followed by the subsequent determination of moxifloxacin MICs with Etest strips.

Effect of gyrA and parC mutations on growth in the presence of moxifloxacin

Three isolates, one (A32) lacking mutations in either gyrA or parC, one (A1069) with a mutation in gyrA, and one (A2180) with mutations in both gyrA and parC, were used to study the effect of gyrA and parC mutations on the growth of A. baumannii isolates in the presence of increasing concentrations of moxifloxacin. Overnight broth cultures of each isolate (107–108 cfu/mL) were spread (100 µL portions of 10-fold dilutions of the culture) on to IsoSensitest agar plates (90 mm diameter) containing moxifloxacin at increasing concentrations (0–128 mg/L). Following incubation at 37°C for 48 h, colony counts (cfu/mL) were plotted against the moxifloxacin concentration in the agar.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Of the 226 A. baumannii isolates examined, 115 (50.9%) were susceptible to ciprofloxacin and 137 (60.6%) were susceptible to moxifloxacin according to the BSAC criteria (MIC of <1.5 mg/L).9 The MICs of ciprofloxacin and moxifloxacin for the E. coli control isolate were <=0.006 mg/L. There was a significant difference in the MICs of ciprofloxacin and moxifloxacin for resistant isolates (Figure 1a). A large proportion (68.5%) of the isolates resistant to ciprofloxacin had an MIC of >=32 mg/L. In contrast, most (87.6%) isolates resistant to moxifloxacin had MICs of 2–16 mg/L. Twenty-two isolates (9.7%) were resistant to ciprofloxacin but remained susceptible to moxifloxacin. None of the isolates examined were resistant to moxifloxacin and susceptible to ciprofloxacin. A. baumannii isolates belonging to the same RAPD genotype and from the same hospital8 often had varying susceptibilities to ciprofloxacin and moxifloxacin, but overall moxifloxacin had better in vitro activity against A. baumannii than ciprofloxacin (Figure 1a). Furthermore, ~20% of ciprofloxacin-resistant isolates remained susceptible in vitro to moxifloxacin according to BSAC breakpoints.9



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Figure 1. (a) MIC (mg/L) distribution of ciprofloxacin and moxifloxacin for nosocomial isolates of A. baumannii. The vertical line indicates the cut-off point used to differentiate fluoroquinolone-susceptible and -resistant isolates.9 (b) Effect of gyrA and parC mutations on growth of A. baumannii isolates in the presence of increasing concentrations of moxifloxacin. A32 is susceptible to ciprofloxacin (MIC 0.38 mg/L) and moxifloxacin (MIC 0.38 mg/L) and has no mutation in either gyrA or parC genes. A1069 is resistant to ciprofloxacin (MIC 12 mg/L) but susceptible to moxifloxacin (MIC 1 mg/L) and has a mutation in gyrA at codon 83 but no mutation in parC. A2180 is resistant to ciprofloxacin (MIC >32 mg/L) and moxifloxacin (MIC 12 mg/L) and has mutations in both gyrA and parC genes.

 
All isolates resistant to ciprofloxacin alone or ciprofloxacin and moxifloxacin (111 in total) had a mutation in the gyrA gene at codon Ser-833 and a ciprofloxacin MIC of 2–>32 mg/L. However, isolates which just possessed this mutation had a moxifloxacin MIC of 0.25–1 mg/L. Only when isolates possessed an additional mutation at codon Ser-80 of the parC gene (see below) did the moxifloxacin MIC become >=2 mg/L. A previous study similarly reported that a double mutation in the gyrA and parC genes was necessary for high-level ciprofloxacin resistance in A. baumannii.6 However, a single mutation in gyrA was sufficient for a clinically significant level of ciprofloxacin resistance.

Of 89 isolates resistant to ciprofloxacin and moxifloxacin, 87 had a mutation in the parC gene at codon Ser-80.4 Two isolates lacked this mutation, despite being resistant to ciprofloxacin and moxifloxacin. These isolates may possess an alternative mutation in a neighbouring codon of parC, or could have a mutation in parE.6,7 Alternatively, these isolates could have altered permeability to fluoroquinolones or an efflux-pump mechanism.2,5,6 Nevertheless, the results from this study suggest that mutations in both gyrA and parC genes are normally required for significant moxifloxacin resistance to develop in A. baumannii. Figure 1(b) illustrates the effect of different combinations of gyrA and parC mutations on the growth of A. baumannii isolates in the presence of increasing concentrations of moxifloxacin.

Twenty-two isolates that were ciprofloxacin resistant but moxifloxacin susceptible did not have the Ser-80 mutation in ParC. The results of spontaneous single-step mutation studies for each of these 22 isolates are displayed in Table 1. Only one isolate (A1066) did not generate spontaneous mutants at a detectable frequency following selection at moxifloxacin concentrations above the initial MIC (Table 1). Ten isolates generated mutants at up to twice the initial MIC, a further eight isolates produced mutants at four times the initial MIC and the remaining three isolates generated mutants at eight times the initial MIC (Table 1). However, when 73 single-step mutants that grew at the highest antibiotic concentration for each respective isolate were examined for high-level moxifloxacin resistance, none had an MIC of >8 mg/L. Five of the 73 single-step mutants examined had a moxifloxacin MIC of 8 mg/L. The remainder had a moxifloxacin MIC of <=6 mg/L. None of the five mutants with a moxifloxacin MIC of 8 mg/L had the mutation at Ser-80 of ParC. Resistance to moxifloxacin appeared to be unstable in these mutants as all five demonstrated a moxifloxacin MIC of <1.5 mg/L following subculture on moxifloxacin-free agar.


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Table 1. Frequency of single-step mutations to moxifloxacin resistance for 22 isolates of ciprofloxacin-resistant, moxifloxacin-susceptible A. baumannii
 
An interesting finding of the single-step mutation studies was that even ciprofloxacin-resistant isolates failed to yield stable moxifloxacin-resistant mutants at detectable frequencies. Furthermore, the mechanism of resistance in the mutants obtained did not appear to be a ParC mutation. None of the mutants with the highest moxifloxacin MICs had the ParC mutation at Ser-80. Resistance to moxifloxacin seemed to be unstable, since mutants grown on moxifloxacin-free agar regained their susceptibility to moxifloxacin. Hence, the mechanism of resistance in these mutants could be associated with altered permeability or an efflux pump that is induced in the presence of subinhibitory concentrations of moxifloxacin. Overall, it was concluded that moxifloxacin had a greater in vitro activity against A. baumannii than ciprofloxacin, coupled with a low likelihood of the generation of stable resistant mutants.


    Acknowledgements
 
This study was supported by an unrestricted grant from Bayer plc.


    Footnotes
 
* Corresponding author. Tel: +44-115-9709163; Fax: +44-115-9422190; E-mail: Kevin.Towner{at}mail.qmcuh-tr.trent.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Bergogne-Berezin, E. & Towner, K. J. (1996). Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clinical Microbiology Reviews 9, 148–65.[Free Full Text]

2 . Towner, K. J. (1997). Clinical importance and antibiotic resistance of Acinetobacter spp. Journal of Medical Microbiology 46, 721–46.[Abstract]

3 . Vila, J., Ruiz, J., Goni, P. et al. (1995). Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 39, 1201–3.[Abstract]

4 . Vila, J., Ruiz, J., Goni, P. et al. (1997). Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 39, 757–62.[Abstract]

5 . Magnet, S., Courvalin, P. & Lambert, T. (2001). Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrobial Agents and Chemotherapy 45, 3375–80.[Abstract/Free Full Text]

6 . Vila, J., Ribera, A., Marco, F. et al. (2002). Activity of clinafloxacin, compared with six other quinolones, against Acinetobacter baumannii clinical isolates. Journal of Antimicrobial Chemotherapy 49, 471–7.[Abstract/Free Full Text]

7 . Wisplinghoff, H., Decker, M., Haefs, C. et al. (2003). Mutations in gyrA and parC associated with resistance to fluoroquinolones in epidemiologically defined clinical strains of Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 51, 177–80.[Free Full Text]

8 . Spence, R. P., Towner, K. J., Henwood, C. J. et al. (2002). Population structure and antibiotic resistance of Acinetobacter DNA group 2 and 13TU isolates from hospitals in the UK. Journal of Medical Microbiology 51, 1107–12.[Abstract/Free Full Text]

9 . Andrews, J. M. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

10 . MacGowan, A. P. & Wise, R. (2001). Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 17–28.[Abstract/Free Full Text]