Evidence of cross-resistance between ciprofloxacin and non-fluoroquinolones in European Gram-negative clinical isolates

P. G. Higgins1,*, A. C. Fluit2, D. Hafner3, J. Verhoef2 and F.-J. Schmitz1,2

1 Institute for Medical Microbiology and Virology, and 3 Institute of Pharmacology and Clinical Pharmacology, Universitätsklinikum Düsseldorf, Universitätsstraße 1, Geb. 22.21, 40225 Düsseldorf, Germany; 2 Eijkman-Winkler Institute for Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands

Sir,

We report here a study that was undertaken to investigate whether ciprofloxacin resistance in Gram-negative nosocomial pathogens is a surrogate marker for cross-resistance to non-fluoroquinolones. The organisms, Escherichia coli (n = 3315), Pseudomonas aeruginosa (n = 1370), Enterobacter aerogenes (n = 252), Enterobacter cloacae (n = 495), Proteus mirabilis (n = 379), Serratia marcescens (n = 207) and Citrobacter freundii (n = 98) were isolated from European hospitals in 1997–1999 as part of the European SENTRY antimicrobial surveillance programme. MICs and breakpoint values of ciprofloxacin, tetracycline, gentamicin, piperacillin, piperacillin/tazobactam, cefuroxime and ceftazidime were determined by broth microdilution according to NCCLS guidelines.1 The isolates were grouped as either ciprofloxacin susceptible or resistant, and the number and percentage of organisms resistant to the antimicrobials are shown in Table 1. The statistical significance of cross-resistance was calculated by Fisher’s exact test and was corrected using the Bonferroni–Holm procedure.


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Table 1.  Numbers of isolates resistant to six antimicrobials for ciprofloxacin-susceptible and -resistant Gram-negative nosocomial pathogens
 
Ciprofloxacin resistance was most prevalent in E. aerogenes, with 27% of isolates resistant, followed by P. aeruginosa with 24%. E. cloacae and S. marcescens had the lowest incidence of ciprofloxacin resistance, at 5% and 7%, respectively. Amongst the ciprofloxacin-susceptible isolates, piperacillin/tazobactam was the most potent agent, with resistance rates of 0–9%. Piperacillin on its own was less potent with resistance levels of up to 38%. Ciprofloxacin-resistant P. aeruginosa and E. cloacae isolates show more than a six- and three-fold increase in piperacillin resistance, and a seven-fold rise in resistance levels to piperacillin/tazobactam (P < 0.01). The other organisms showed a two- to four-fold increase in resistance to these agents, and S. marcescens exhibited a seven-fold increase in resistance to piperacillin. Ciprofloxacin-resistant P. mirabilis remained 100% susceptible to piperacillin/tazobactam. Gentamicin resistance was also relatively low in the ciprofloxacin-susceptible group, with resistance rates of 1.6–7.1%. These rates increased two- to 14-fold in the ciprofloxacin-resistant group, with E. coli showing the largest increase.

Tetracycline resistance rates increased 16-fold for ciprofloxacin-resistant E. aerogenes (P < 0.05); for the other organisms this increase was a more modest four-fold or less. P. mirabilis isolates are almost universally resistant to tetracycline. Nearly all P. aeruginosa isolates were cefuroxime resistant, irrespective of ciprofloxacin resistance. For the other organisms, cefuroxime resistance levels were less than or equal to seven-fold higher if ciprofloxacin resistant, and S. marcescens was 100% resistant. A similar trend was seen with ceftazidime: a two- to nine-fold increase in resistance associated with ciprofloxacin resistance. Imipenem was tested against P. aeruginosa and resistance levels were six-fold higher in the ciprofloxacin-resistant population (P < 0.01). No significant cross-resistance was seen with C. freundii.

These data show that the relationship between ciprofloxacin resistance and resistance to unrelated drugs was statistically significant. There exist interspecies differences on the effects of ciprofloxacin resistance, for example gentamicin/ciprofloxacin resistance in E. aerogenes is not statistically significant compared with E. coli (P < 0.001). However, ciprofloxacin resistance can be used as a general surrogate marker of multidrug resistance.

The resistance mechanisms of the isolates were not investigated in this study, nor was the relatedness of isolates within a species; therefore, the process by which an organism goes from susceptible to multidrug resistant is open to speculation. Ciprofloxacin resistance probably followed multidrug resistance, given that resistance levels are low in the ciprofloxacin-susceptible group. Indeed, ciprofloxacin therapy might have been used when other therapeutic options were compromised because of resistance. However, ciprofloxacin therapy has been shown to select for multidrug resistance by causing mutational changes in efflux regulatory genes in P. aeruginosa.2,3 Efflux systems are near-ubiquitous in microorganisms, and their effect on a broad range of substrates either confers resistance or allows survival until resistance genes can be acquired. Efflux on its own favours clonal spread of an organism but does not allow for horizontal transfer of resistance.

Another route to multidrug resistance is mediated through the acquisition and dissemination of resistance determinants. These resistance genes are found on integrons and are disseminated through transposons and plasmids. Genes encoding resistance to extended-spectrum ß-lactams, aminoglycosides, chloramphenicol, co-trimoxazole, streptomycin, sulfamethoxazole and quaternary ammonium compounds have all been found on integrons in the Enterobacteriaceae. Leverstein-Van Hall et al.4 found integrons with resistance genes against drugs that had been used rarely, if ever, in the past 20 years, indicating the persistence and stability of these genes without selective pressure. Jones et al.5 found that integrons carrying aminoglycoside-modifying enzymes are widespread throughout Europe. Transfer of resistance from a Klebsiella donor to an E. coli recipient showed that the integron was on a plasmid. Martinez-Freijo et al.6 found that integron-positive isolates were statistically more likely to be multidrug resistant than integron-negative isolates. Leverstein-Van Hall et al.4 also showed that integrons are associated with multidrug-resistant Enterobacteriaceae. These were found to be involved in horizontal transfer, as the same integrons were found on different species that had colonized patients. These integrons were transferred experimentally at high frequency.

The threat of multidrug resistance and the need for surveillance is apparent. A more detailed epidemiological study and an investigation into the causes of resistance, in particular the horizontal spread of resistance genes in nosocomial pathogens, is needed to counter the threat posed by these organisms.

Footnotes

* Corresponding author. Tel/Fax: +49-21-3272040; E-mail: Paul.Higgins{at}uni-duesseldorf.de Back

References

1 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

2 . Nakano, M., Yasuda, M., Yokoi, S., Takahashi, Y., Ishihara, S. & Deguchi, T. (2001). In vivo selection of Pseudomonas aeruginosa with decreased susceptibilities to fluoroquinolones during fluoroquinolone treatment of urinary tract infection. Urology 58, 125–8.[ISI][Medline]

3 . Le Thomas, I., Couetdic, G., Clermont, O., Brahimi, N., Plesiat, P. & Bingen, E. (2001). In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy. Journal of Antimicrobial Chemotherapy 48, 553–5.[Abstract/Free Full Text]

4 . Leverstein-Van Hall, M. A., Box, A. T. A., Blok, H. E. M., Paauw, A., Fluit, A. D. & Verhoef, J. (2002). Evidence of extensive interspecies transfer of integron-mediated antimicrobial resistance genes among multiresistant Enterobacteriaceae in a clinical setting. Journal of Infectious Diseases 186, 49–56.[ISI][Medline]

5 . Jones, M. E., Peters, E., Weersink, A. M., Fluit, A. & Verhoef, J. (1997). Widespread occurrence of integrons causing multiple antibiotic resistance in bacteria. Lancet 349, 1742–3.[ISI][Medline]

6 . Martinez-Freijo, P., Fluit, A. C., Schmitz, F.-J., Grek, V. S. C., Verhoef, J. & Jones, M. E. (1998). Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds. Journal of Antimicrobial Chemotherapy 42, 689–96.[Abstract]





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