Relationship between haemolysis production and resistance to fluoroquinolones among clinical isolates of Escherichia coli

Luis Martínez-Martíneza,b,*, Felipe Fernándeza and Evelio J. Pereaa,b

a Department of Microbiology, University Hospital Virgen Macarena; b School of Medicine, Seville, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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 ({alpha}-haemolysin), a cell-bound protein (ß-haemolysin) and a haemolysin expressed by nalidixic acid-resistant mutants ({gamma}-haemolysin). 3,4 {gamma}-Haemolysin cannot haemolyse human or rabbit red blood cells, unlike {alpha}- and ß-haemolysins.4 {alpha}-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 {alpha}-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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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 {gamma}-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 {chi}2 test and (in the case of gentamicin) Fisher's exact test. Differences were considered significant when P was <0.05.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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 {alpha}-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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Table. Resistance to eight antimicrobial agents among 57 haemolytic and 150 non-hemolytic clinical isolates of E. coli
 
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 {gamma}-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.


    Notes
 
* 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 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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]

3 . Cavalieri, S. J., Bohach, G. A. & Snyder, I. S. (1984). Escherichia coli {alpha}-hemolysin: characteristics and probable role in pathogenicity. Microbiological Reviews 48, 326–43.[ISI]

4 . Walton, J. R. & Smith, D. H. (1969). New hemolysin ({gamma}) produced by Escherichia coli. Journal of Bacteriology 98, 304–5.[ISI][Medline]

5 . Welch, R. A. & Pellet, S. (1988). Transcriptional organization of the Escherichia coli hemolysin genes. Journal of Bacteriology 170, 1622–30.[ISI][Medline]

6 . Wandersman, C. & Delepelaire, P. (1990).TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proceedings of the National Academy of Sciences of the USA 87, 4776–80.[Abstract]

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.[ISI][Medline]

9 . Bauer, M. E. & Welch, R. A. (1997). Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin. Infection and Immunity 65, 2218–24.[Abstract]

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