Pyelonephritis caused by multiple clones of Escherichia coli, susceptible and resistant to co-amoxiclav, after a 45 day course of co-amoxiclav

Véronique Leflon-Guibouta, Stéphane Bonacorsib, Olivier Clermontb, Géraldine Ternata, Beate Heyma and Marie-Hélène Nicolas-Chanoinea,*

a Service de Microbiologie, Hôpital Ambroise Paré (AP-HP), Faculté de Médecine Paris-Ouest, Université Paris V, Boulogne; b Service de Microbiologie, Laboratoire d'Etudes de Génétique Bactérienne dans les Infections de l'Enfant EA 3105, Hôpital Robert Debré, Paris, France


    Abstract
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 Abstract
 Introduction
 Patient and methods
 Results and discussion
 References
 
Objectives: We studied the clonal relatedness, in terms of phylogenetic group, virulence factors and co-amoxiclav resistance, of different Escherichia coli isolates obtained from blood and urine of a patient who had taken a 45 day course of co-amoxiclav.

Patients and methods: The isolates were typed by molecular methods, and the virulence and resistance genes studied by PCR.

Results: The four phenotypically different E. coli isolates were classified into four clones and two phylogenetic groups (B2 and A), and displayed different virulence gene patterns. The two isolates resistant to co-amoxiclav were phylogenetically and clonally different, and harboured two different blaTEM genes.

Conclusion: These two blaTEM genes did not derive from each other following a mutant selection process in vivo.


    Introduction
 Top
 Abstract
 Introduction
 Patient and methods
 Results and discussion
 References
 
Between 1980 and 1995, a series of reports showed that polymicrobial bacteraemia accounted for 6–12% of all bacteraemias.1–3 This type of serious infection most commonly resulted from genitourinary and gastrointestinal infections, and often occurred in elderly people with significant underlying diseases, notably malignancies, and those undergoing cancer chemotherapy.1–3 Gram-negative aerobic bacilli were frequently implicated, particularly Escherichia coli and Klebsiella spp., when the source of bacteraemia was community-acquired urosepsis. Bacteraemias caused by multiple isolates of the same species were very rarely reported. One case of multiple isolates of E. coli from the blood and urine of a 60-year-old woman was described by Johnson et al.4 These authors were able to differentiate the E. coli isolates on the basis of biotype, antimicrobial susceptibility pattern and plasmid profile. Nevertheless, they found that the different isolates corresponded to the same strain on the basis of DNA fingerprints.

We report here a case of pyelonephritis in which multiple E. coli isolates were obtained from the blood (n = 3) and urine (n = 2) of a female patient 2 weeks after finishing a 45 day course of co-amoxiclav. The two urinary isolates displayed two different ß-lactam resistance phenotypes, but both were resistant to co-amoxiclav, whereas only one of the three blood isolates was resistant to this antibiotic. This infection was cured but was followed 1 month later by a second urinary tract infection caused by a single E. coli resistant to co-amoxiclav. As all the E. coli isolates from the patient were available, molecular analyses were carried out to define (i) whether these isolates were clonally related, (ii) whether they had the same virulence factor gene pattern and belonged to the same phylogenetic group, and (iii) whether the TEM ß-lactamases produced by the different co-amoxiclav-resistant isolates derived from each other following in vivo mutant selection by co-amoxiclav treatment.


    Patient and methods
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 Abstract
 Introduction
 Patient and methods
 Results and discussion
 References
 
Patient

Mrs K. (58 years old), was hospitalized in the middle of September 1997 in the A. Paré Hospital for chemotherapy of recurring breast cancer that was originally diagnosed in 1978. This relapse presented as oedema of the arm and was initially mistaken for lymphangitis, and so was treated with a 45 day course of co-amoxiclav, which the patient had finished 2 weeks before being hospitalized. During this hospitalization, she developed pyelonephritis caused by E. coli, the isolates of which included several colonies that were morphologically different and had different antibiotic resistance patterns (TableGo). The patient was given a 5 day course of intravenous ceftriaxone (1 g/day) and was discharged from the hospital on day 9. One month later, which corresponded to 20 days after the second cancer chemotherapy treatment (5-fluorouracil-docetaxel), she was hospitalized again for pyelonephritis caused by an E. coli resistant to co-amoxiclav, with an antibiotic resistance pattern identical to one of the two isolates obtained from urine sample and to one of the three isolates obtained from the bloodstream during the first infection (TableGo). Faced with this second infection, cured with a 4 day course of ceftriaxone followed by a 7 day course of oral ciprofloxacin (500 mg bd), the third cancer chemotherapy treatment was postponed and various investigations were performed, notably a urinary tract and kidney ultrasound scan. No abnormalities were found. While the patient was on ciprofloxacin, we looked for co-amoxiclav-resistant E. coli in faecal samples, but no Gram-negative bacilli could be grown.


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Table. Clinical and molecular characterization of the six E. coli isolates
 
Bacterial identification and antibiotic susceptibility

Bacterial identification was carried out with the API system (bioMérieux, Marcy l'Étoile, France). The antibiotic susceptibility of E. coli isolates was determined using the agar disc diffusion method, and interpreted according to the recommendations of the French Committee of Antibiogrammes.5 The following antibiotics were tested: amoxicillin, co-amoxiclav, cefalothin, cefuroxime, cefotaxime, kanamycin, gentamicin, chloramphenicol, tetracycline, sulphonamides, trimethoprim, nalidixic acid and pefloxacin.

Molecular analysis

The clonal relatedness of the different E. coli isolates was assessed by DNA profile obtained by the random amplified polymorphic DNA (RAPD) typing system as described previously.6 The two primers singly used for RAPD typing were HLWL74: 5'-ACGTATCTGC-3', and A3: 5'-TGGACCCTGC-3'. The phylogenetic group to which the E. coli isolates belonged was determined by using a pre viously described PCR-based method.7 The presence of seven putative virulence factor genes (TableGo), characteristic of extra-intestinal pathogenic E. coli, was determined by PCR using primers and conditions described previously.8,9 The underlying mechanism of ß-lactam resistance was defined by using amplification reactions specific for blaTEM genes and by sequencing, as described previously.10


    Results and discussion
 Top
 Abstract
 Introduction
 Patient and methods
 Results and discussion
 References
 
As indicated in the FigureGo, the six E. coli isolates were classified into four groups on the basis of the RAPD profiles produced with the primers HLWL74 and A3. Group I and group II each included only one strain, K1 (urine) and K5 (blood), respectively, whereas group III included three strains, namely K2 (urine), K4 (blood) and K6 (urine) (Figure, aGo).



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Figure. RAPD profiles of the six E. coli strains. (a) RAPD profiles obtained with the primer HLWL74 for the six isolates. Strains K1 and K5 differ from the four other strains by several bands. Strains K2, K4 and K6 display an identical profile, whereas K3 can be distinguished from these three strains by three bands, two of which have a low intensity. (b) RAPD profiles obtained with the primer A3 for strains K3, K6, K4 and K2. Strains K6, K4 and K2 display an identical profile, whereas K3 differs from these three strains by three high intensity bands. M, weight marker; NC, negative control.

 
Group IV, which included only strain K3 (blood), was more readily distinguished from group III using the primer A3 than using the primer HLWL74 (FigureGo).

The three strains of group III and the group IV strain belonged to phylogenetic group A, the group characteristic of commensal E. coli, whereas the group I and II strains belonged to phylogenetic group B2, characteristic of extra-intestinal pathogenic E. coli (TableGo).9 Three virulence factor genes (aer, fyuA and irp2), involved in iron acquisition, were present in all strains, regardless of the clonal and the phylogenetic group (TableGo). Additional genes, notably pap genes, were detected in the two strains belonging to phylogenetic group B2, one of them having all the genes tested including toxin-encoding genes (hly and cnf1) (TableGo). Only one strain of phylogenetic group A (blood strain K3) also possessed the papC gene (TableGo).

blaTEM-specific PCR, which was systematically applied to the DNA template extracted from the six strains, was positive for strains K1, K2, K4 and K6 and negative for strains K3 and K5. Sequencing of the four amplified products showed that strains K2, K4 and K6 harboured an identical blaTEM gene, which resembled the blaTEM-1F gene except for an A407C mutation leading to the substitution Met-69->Leu at the origin of the inhibitor-resistant TEM-33. Strain K1 was shown to harbour a blaTEM gene identical to the blaTEM-1A except for the C32T mutation, which resulted in the creation of the strong promoter Pa/Pb. Such a promoter, which had been found previously in different blaTEM genes but not, until this study, upstream of the blaTEM-1A gene, supports the possibility of the hyperproduction of the TEM-1 enzyme resulting in co-amoxiclav resistance of strain K1.

Our extensive molecular study of the six isolates allowed us to show that this patient had an initial episode of pyelonephritis caused by four different strains of E. coli. These strains were not equally pathogenic. Indeed, two of them belonged to the phylogenetic group B2, a group from which uropathogenic E. coli are chiefly reported.9 The two other strains, although they were apparently less virulent since they belonged to phylogenetic group A and had no PapG adhesin, were also isolated from blood. However, these two latter strains harboured the two iron uptake systems (aer and fyuA, irp2), that are mainly encountered in strains responsible for bacteraemia. The second relevant point shown by this study is that the second episode of pyelonephritis, which occurred 1 month after initial successfully treated infection, was caused by one of the four original strains, strain K6, which belonged to the apparently least virulent clone (phylogenetic group A having fewest virulence factor genes). However, this strain was resistant to co-amoxiclav through the production of an inhibitor-resistant TEM (IRT) enzyme, and so was resistant to an antibiotic that the patient had taken for 45 days until 2 weeks before the first infection. Such resistance was also found in another strain from the first infection episode, K1, which was apparently more virulent than strain K6, as it belonged to the phylogenetic group B2 and had all seven virulence factor genes tested. Why strain K6 had more capacity than strain K1 to cause the second episode of pyelonephritis remains unexplained. Nevertheless some hypotheses can be formed. The mechanism by which strain K6 was resistant to co-amoxiclav (IRT) is more stable and efficient than TEM-1 hyperproduction displayed by strain K1. Thus, the E. coli clone producing the IRT enzyme, which was probably selected like the TEM-hyperproducing clone during co-amoxiclav treament, could have reached a higher concentration in the digestive tract than the TEM-hyperproducing clone, and thus carriage could have persisted in spite of ceftriaxone given for the first pyelonephritis episode. We were not able to prove this fact, as there were no Gram-negative bacilli in the patient's faeces when we searched for digestive co-amoxiclav-resistant E. coli isolates while the patient was taking ciprofloxacin (which is known to reduce aerobic bacteria in the digestive tract). The advantage conferred by the IRT enzyme with regard to resistance to co-amoxiclav and the compromised status of the patient following repeated cancer chemotherapy suggest why a clone of the phylogenetic group A (from which commensal E. coli derive) might have been responsible for a second episode of pyelonephritis in this patient, who had no urinary tract abnormality.

The last significant point shown by our study is that the gene encoding IRT (blaTEM-33F) was not a derivative of the gene encoding TEM (blaTEM-1A) following an in vivo mutant selection process. Indeed, these two genes differed from each other by seven mutations and, moreover, were expressed by two strains belonging to two different clones.


    Notes
 
* Correspondence address. Service de Microbiologie, Hôpital A. Paré, 9 avenue Charles de Gaulle, 92100 Boulogne-Billancourt, France. Tel: +33-1-49-09-55-40; Fax: +33-1-49-09-59-21; E-mail: marie-helene.nicolas-chanoine{at}apr.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Patient and methods
 Results and discussion
 References
 
1 . Kiani, D., Quinn, E. L., Burch, K. H., Madhavan, T., Saravolatz, L. D. & Neblett, T. R. (1979). The increasing importance of polymicrobial bacteremia. Journal of the American Medical Association 242, 1044–7.[Abstract]

2 . Cooper, G. S., Havlir, D. S., Shlaes, D. M. & Salata, R. A. (1990). Polymicrobial bacteremia in the late 1980s: predictors of outcome and review of literature. Medicine (Baltimore) 69, 114–23.[ISI][Medline]

3 . Whitelaw, D. A., Rayner, B. L. & Willcox, P. A. (1992). Community-acquired bacteremia in the elderly: a prospective study of 121 cases. Journal of the American Geriatrics Society 40, 996–1000.[ISI][Medline]

4 . Johnson, J. R., Moseley, S. L., Coyle, M. B. & Stamm, W. E. (1992). Success of DNA fingerprinting after failure of biotyping, antimicrobial susceptibility testing, and plasmid analysis to reveal clonality of multiple blood and urine isolates from a patient with Escherichia coli urosepsis. Diagnostic Microbiology & Infectious Disease 15, 399–405.[ISI][Medline]

5 . Anonymous. (1996). Statement 1996 Ca-SFM Zone sizes and MIC breakpoints for non-fastidious organisms. Clinical Microbiology and Infection 2, S46–7.[Medline]

6 . Bingen, E., Bedu, A., Brahimi, N. & Aujard, Y. (1995). Use of molecular analysis in pathophysiological investigation of late-onset neonatal Escherichia coli meningitis. Journal of Clinical Microbiology 33, 3074–6.[Abstract]

7 . Clermont, O., Bonacorsi, S. & Bingen, E. (2000). Rapid and simple determination of the Escherichia coli phylogenetic group. Applied Environmental Microbiology 66, 4555–8.[Abstract/Free Full Text]

8 . Bingen, E., Picard, B., Brahimi, N., Mathy, S., Desjardins, P., Elion, J. et al. (1998). Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains. Journal of Infectious Diseases 177, 642–50.[ISI][Medline]

9 . Johnson, J. R. & Stell, A. L. (2000). Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. Journal of Infectious Diseases 181, 261–72.[ISI][Medline]

10 . Leflon-Guibout, V., Speldooren, V., Heym, B. & Nicolas-Chanoine, M.-H. (2000). Epidemiological survey of amoxicillin-clavulanate resistance and corresponding molecular mechanisms in Escherichia coli isolates in France: new genetic features of blaTEM genes. Antimicrobial Agents and Chemotherapy 44, 2709–14.[Abstract/Free Full Text]

Received 29 May 2001; returned 1 October 2001; revised 7 November 2001; accepted 20 November 2001





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