Journal of Antimicrobial Chemotherapy (1999) 43, 277-279
© 1999 The British Society for Antimicrobial Chemotherapy
Brief reports |
Relationship between haemolysis production and resistance to fluoroquinolones among clinical isolates of Escherichia coli
a Department of Microbiology, University Hospital Virgen Macarena; b School of Medicine, Seville, Spain
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Abstract |
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The activities of ampicillin, amoxycillin-clavulanic acid, gentamicin, tetracycline, nalidixic acid, ciprofloxacin, pefloxacin and trovafloxacin against 207 consecutive clinical isolates of Escherichia coli were determined. Fifty-six (27.3%) isolates were haemolytic. The percentages of resistance to quinolones and tetracycline, but not to other agents, among haemolytic isolates were significantly lower (P < 0.05) than among non-haemolytic isolates. Ciprofloxacin-resistant mutants obtained from ciprofloxacin-susceptible haemolytic isolates still produced haemolysis. It is concluded that most quinolone-resistant clinical isolates of E. coli are non-haemolytic, although haemolysis is produced by quinolone-resistant mutants derived from haemolytic quinolone-susceptible strains.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Resistance to fluoroquinolones in Escherichia coli is an increasing problem in Spain and other countries. 1 Several mechanisms are known to determine this resistance in E. coli, including mutations in the topoisomerase (II and IV) genes, and decreased accumulation because of outer membrane alterations and/or the expression of efflux pumps. 2
E. coli can produce several types of haemolysin, including an extracellular protein
(
-haemolysin), a cell-bound protein (ß-haemolysin) and a haemolysin expressed by
nalidixic acid-resistant mutants (
-haemolysin).
3,4 -Haemolysin
cannot haemolyse human or rabbit red blood cells, unlike - and ß-haemolysins.4 -Haemolysin is known to be a product of the hlyCABD operon.
5 The outer membrane protein TolC is also required for
haemolysin export to the extracellular medium.
6 -Haemolysin is a virulence factor in strains causing
different extra-intestinal infections. The protein can induce osmotic lysis of erythrocytes because
of its pore-forming activity, and is cytotoxic to several types of human cell.
3
While determining antimicrobial susceptibility in our clinical laboratory we have often observed that E. coli strains resistant to quinolones were non-haemolytic. This study was undertaken to evaluate this observation and to determine whether quinolone-resistant mutants of haemolytic quinolone-susceptible E. coli strains can still produce haemolysin.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Bacterial strains
Two hundred and seven consecutive clinical isolates of E. coli obtained from different patients referred to the clinical laboratory of the University Hospital Virgen Macarena, Seville, Spain in September and October 1996 were evaluated. Organisms were cultured from urine (86%), peritoneal fluid (5%), blood culture (4%), wound exudate (4%) and other sites (1%). Identification was performed with the WalkAway system (MicroScan, Dade, Sacramento, CA, USA), with panel types Urine-Combo 6I (urine isolates) and Neg-Combo 6I (organisms from other samples), according to the manufacturer's instructions. Organisms were maintained in tryptic soy broth containing 10% glycerol at -30°C until used for further studies.
Susceptibility testing
Preliminary susceptibility testing was performed with the same panels used for bacterial identification. Definitive susceptibility testing was performed by microdilution, according to NCCLS guidelines. 7 The following antimicrobial agents were studied: ampicillin (Sigma, Madrid, Spain), amoxycillin (Sigma) plus clavulanic acid (SmithKline Beecham, Madrid, Spain) at a fixed concentration of 2 mg/L, gentamicin (Sigma), tetracycline (Sigma), nalidixic acid (Sigma), ciprofloxacin (Sigma), pefloxacin (Rhône-Poulenc, Antony, France) and trovafloxacin (Pfizer, Groton, CT, USA). Etest strips (AB Biodisk, Solna, Sweden) were also used to determine MICs of tetracycline for 22 isolates (including nine haemolytic isolates) because of discrepancies between the WalkAway and reference microdilution methods. The values determined by reference microdilution were considered definitive as they agreed with those determined with Etest strips.
Organisms were considered resistant to the antimicrobial agents evaluated when the
corresponding MICs (mg/L) were 
16 (ampicillin), 16/2 (amoxycillin-clavulanic
acid),
8 (gentamicin, tetracycline, pefloxacin), 32 (nalidixic acid) and 2
(ciprofloxacin
and trovafloxacin). These breakpoints allowed comparison of susceptible versus non-susceptible
(either intermediate or resistant) isolates, according to NCCLS guidelines.
7 Trovafloxacin is not included in the NCCLS document, so
the breakpoint used was the same as that defined for ciprofloxacin. For amoxycillin-clavulanic
acid, the breakpoint defined by the NCCLS is 16/8, but we used clavulanate in a fixed
concentration of 2 mg/L .
In-vitro selection of quinolone-resistant mutants
Ciprofloxacin-resistant mutants were selected from two haemolytic clinical isolates for which the MIC of ciprofloxacin was 0.015 mg/L. A log phase culture in Mueller-Hinton broth was inoculated on Mueller-Hinton agar plates containing 5% sheep blood agar and 1, 2 or 4 x MIC of ciprofloxacin. Plates were incubated at 37°C for 48 h. Mutants with increased resistance to ciprofloxacin were obtained after repeated subculture under the same conditions. Mutants were subcultured on antibiotic-free medium and stored for MIC and haemolysis determination.
Determination of haemolytic activity
An organism was considered haemolytic when a clear halo was observed around isolated
colonies after overnight incubation. The organisms were considered -haemolysin producers
when haemolysis was observed on Columbia agar base containing sheep blood (5%,
BioMérieux, Marcy l' Etoile, France) but not when containing human blood
(5%).
Statistical methods
The statistical significance of differences in resistance to antimicrobial agents between
haemolytic and non-haemolytic isolates was tested using the 
2 test and (in
the case of gentamicin) Fisher's exact test. Differences were considered significant when P was <0.05.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Overall, the percentages of resistance among the 207 isolates evaluated were: 61.9% for ampicillin, 16.4% for amoxycillin-clavulanic acid, 3.6% for gentamicin, 36.8% for tetracycline, 36.2% for nalidixic acid, 21.5% for ciprofloxacin, 23.7% for pefloxacin and 10.8% for trovafloxacin. It should be noted that the figure for resistance to amoxycillin-clavulanic acid includes (at least) organisms corresponding to both the intermediate and resistant categories of the NCCLS, and for only 5.8% of the organisms was the MIC >32/2 mg/L. The rates of quinolone resistance reported here are similar to those presented in other studies from Spain, and higher than those of a previous study in our institution, 1 confirming the tendency to increased quinolone resistance during recent years.
Fifty-seven (27.5%) isolates were haemolytic on both sheep and human blood agar, suggesting
that these organisms produce -haemolysin. The percentages of resistance to quinolones
(nalidixic acid, ciprofloxacin, pefloxacin and trovafloxacin) and to tetracycline, but not to other
agents, were significantly higher (P < 0.05) among non-haemolytic isolates than
among haemolytic isolates (Table). Hariharan et al.
8 have shown that resistance to co-trimoxazole, neomycin
and tetracycline in E. coli strains isolated from piglets with diarrhoea was less frequent
among strains producing heat-labile enterotoxin (LT) and haemolysin than among those lacking
both factors, while the resistance to gentamicin was more frequent in LT-haemolysin producers
than among LT-haemolysin non-producers. The relationship between haemolysin production and
resistance to enrofloxacin could not be evaluated, as all strains were susceptible to enrofloxacin.
Our data for tetracycline are similar to those obtained by these authors, but we have not found a
significant difference in resistance to gentamicin among haemolytic and non-haemolytic isolates.
In fact, the few gentamicin-resistant strains in our study were non-haemolytic.
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The mechanisms responsible for the predominance of non-haemolytic E. coli strains among those expressing resistance to quinolones are unknown. Ciprofloxacin-resistant mutants (MIC of ciprofloxacin ranging from 0.03 to > 32 mg/L) derived in vitro from haemolytic-susceptible isolates still produced haemolysis. These mutants are not related to the previously described
-haemolysin producers (selected in the presence of nalidixic acid) as
they still haemolysed human erythrocytes. It is not known if resistance to quinolones and loss of
haemolysis are caused by common or related mechanisms or if these phenotypes are derived
from
independent mutations. Most clinical isolates of E. coliresistant to quinolones are gyrA mutants,
2 and it is possible that altered supercoiling of DNA in these
mutants may affect the expression of genes involved in haemolysis production. Another
possibility is the existence of pleiotropic mutations in quinolone-resistant E. coli strains
that may interfere with the expression or activity of haemolysin, as has recently been reported for
mutations affecting the genes involved in lipopolysaccharide synthesis.
9 Fluoroquinolone resistance in clinical isolates and laboratory-derived mutants of E. coli are frequently associated with decreased expression of type 1 fimbriae, another virulence factor of E. coli. 10 Our study shows that haemolysin is also less frequently produced by quinolone-resistant clinical isolates of E. coli. It is possible that resistance to quinolones has an indirect cost in terms of decreased bacterial virulence. Further studies are needed to test this hypothesis.
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Notes |
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* Correspondence address. Department of Microbiology, School of Medicine, Apdo 914, 41080 Seville, Spain. Tel: +34-95-4557448; Fax:+34-95-4377413; E-mail: lmartin{at}cica.es
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Martínez-Martínez, L., Suárez, A. I., Carranza, R. & Perea, E. J. (1993). Resistencia a ciprofloxacino en bacilos gram negativos. Aspectos epidemiológicos. Enfermedades Infecciosas y Microbiología Clínica 11, 434–8.
2 . Everett, M. J., Fang Jin, Y., Ricci, V. & Piddock, L. J. V. (1996). Contribution of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from human and animals. Antimicrobial Agents and Chemotherapy 40, 2380–6.[Abstract]
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7 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A3. NCCLS, Wayne, PA.
8 . Hariharan, H., Heaney, S., Bryenton, J. & Daley, J. (1992). Observations on production of hemolysin, heat-labile enterotoxin and antimicrobial drug resistance among enterotoxigenic Escherichia coli from pigs. Comparative Immunology, Microbiology and Infectious Diseases 15, 229–34.[Web of Science][Medline]
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10 . Bagel, S., Heisig, P. & Wiedemann, B. (1997). Fluoroquinolone resistance of Escherichia coli frequently is associated with decreased expression of type 1 fimbriae. In Program and Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy. Toronto. Abstract C-37, p. 52. American Society for Microbiology, Washington, DC.
Received 25 March 1998; returned 14 May 1998; revised 17 June 1998; accepted 17 September 1998
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