Integrons and antibiotic resistance in phylogenetic group B2 Escherichia coli.
David Skurnik,1 Sonia Lacheeb,1 Claire Bernede,2 Arnaud Le Menac’h,3 Sandrine Elbaz,1
Jacqueline Mohler,1 Erick Denamur,4 Antoine Andremont,1 Raymond Ruimy1
1
EA3964 Résistance Bactérienne in Vivo, Faculté de Médecine, Université Paris 7 and
Groupe Hospitalier Bichat-Claude Bernard APHP, 75018 Paris, France
2
CeRBEP, Institut Pasteur/ INSERM U657, Paris, France
3
INSERM U444, Paris, France
4
INSERM U722 and Université Paris 7 Denis Diderot
Running title: Integrons and antibiotic resistance in B2 E.coli
Corresponding author:
Raymond Ruimy M.D., Ph.D.
Groupe Hospitalier Bichat-Claude Bernard
Laboratoire de Bactériologie
46, Rue Henri-Huchard
75018 Paris
France
Tel. 33 1 40 25 85 05 / Fax. 33 1 40 25 85 81
e-mail: raymond.ruimy@bch.ap-hop-paris.fr
1
Abstract :
In commensal Escherichia coli isolates, the prevalence of phylogenetic B2 strains differed
according the host but B2 strains tend to carry less integron than the others. B2 strains are also
more sensitive to antibiotics than those of the others groups, especially to quinolones. To have
a better understanding of the relationships between antibiotic resistance, integron, and
phylogenetic groups, we compared the phylogenetic grouping and the content of integrons in
3 selected groups of UTI E. coli isolates (G1, G2, G3), known to comprise a high rate of B2
strains, isolated from patients hospitalized in a French university hospital. The isolates were
fully susceptible to antibiotics for the G1 strains, resistant to amoxicillin and cotrimoxazol for
the G2 strains, and to amoxicillin, cotrimoxazol and nalidixic acid for the G3 strains. Patients
of the 3 groups were matched for age and sex and to eliminate replicates, all isolates were
typed using enterobacterial repetitive intergenic consensus-PCR. The prevalence of
phylogenetic group B2 was significantly lower in the most resistant G3 strains than in
susceptible G1 strains (22.6% versus 57.8, p<0.001) and G2 strains (22.6 versus 50%,
p<0.01). By contrast, the prevalence of phylogenetic group B2 isolates was not significantly
different in G1 and G2 strains. The prevalence of integrons was nil in G1 isolates but very
high in G2 isolates (94.3%) and G3 (87.5%) isolates, suggesting that in environments with
strong antibiotic selective pressure, like in a university hospital, integrons are highly prevalent
in clinical resistant strains and widespread whatever the phylogenetic group.
2
Introduction
The emergence of antibiotic resistance in bacteria is the result of complex interactions
between the environment of the strain, including the exposure to antibiotics, the genomic
constraints and the fitness of the strain. Escherichia coli is particularly well suited to study
such interactions as it is a widespread commensal of gut of vertebrates, including human, that
is inevitably excreted in the external environment. It is also a major pathogen involved in
intestinal and extraintestinal diseases. During these various life styles, E. coli is subjected to
very various selective pressures. Population genetic studies have shown that the species have
a mainly clonal population structure (6,31) with 4 main phylogenetic groups (A, B1, B2 and
D) (14), strains of the 4 different groups differing in their phenotypic and genotypic
characteristics. Strains of the B2, and at a lesser extend D, phylogenetic groups are
predominant among extraintestinal isolates (17,30) whereas differences in prevalence of the 4
phylogenetic groups in commensal strains are observed according to the strain host (7,8,10).
By studying commensal E. coli strains from various hosts exposed to various humans
and/or human activities, we were able to show an increased in resistance linked to human
exposure, probably related to increasing antibiotic selective pressure (33). Furthermore, the
previously reported link between integron (a highly efficient genetic tool for the expression
and spread of antibiotic resistance) prevalence and antibiotic resistance was present in the
strains isolated from pets and farm animals, but not in resistant strains from wild animals,
which had less contact with humans. This suggests that the presence of integrons may
constitute a process of genomic adaptation to environments when antimicrobial pressure
exceeds a certain threshold. In addition to the environment of the host, the genetic background
of the strain seems also to be involved in the emergence of resistance. B2 group strains appear
more sensitive to antibiotics than strains of the other phylogenetic groups (16,29), especially
3
to quinolones (18,21,23,28,34,36). Likely, in human community commensal environment,
strains from the B2 group tended to carry fewer integrons than the others (32).
To have a better understanding of the relationships between resistance, integron,
phylogenetic groups and host environment, we selected, for the present study, three groups of
human urinary tract infection (UTI) E. coli isolates, which are known to comprise a high rate
of B2 strains (17). The first included isolates susceptible to amoxicillin, cotrimoxazol and
quinolone, the second, isolates resistant to amoxicillin and cotrimoxazol but sensitive to
quinolones, and the third, isolates resistant to these three antibiotics. The patients of the 3
groups were matched for age and sex to avoid potential bias linked to the phylogenetic
distribution of E. coli related to these characteristics (11). We then determined and compared
their integron content and phylogenetic grouping.
4
Materials and methods
Bacterial isolates. The strains were selected among the UTI E. coli isolated from patients
hospitalized in the Bichat-Claude Bernard Hospital in Paris between January 2005 and July
2006. Isolates were identified as E. coli using API 20E strips (API, La Balme-les-Grottes,
France) and tested for antibiotic susceptibility by the disk diffusion technique, as previously
described (http://www.sfm.asso.fr). UTI was defined as urine cultures yielding E. coli in pure
culture in counts 105 CFU/ml, with >104 leukocytes per ml. Strains from patients with
urinary catheters were excluded. Three groups of 66 strains each were included: the first (G1)
comprised strains susceptible to amoxicillin (AMX), cotrimoxazol (TMP-SMZ) and nalidixic
acid (NA), the second (G2) of strains resistant to TMP-SMZ and AMX but susceptible to NA,
and the third (G3), of strains resistant to TMP-SMZ, AMX and NA. The 3 groups of patients
from whom the 3 groups of 66 strains each were isolated were matched for age (range: 20 to
85 years) and sex (16 males and 50 females).
DNA Extraction. DNA extraction was performed using MagnaPure LC (Roche, Mannheim,
Germany), following the manufacturer’s recommendations. DNA was quantified at an optical
density of 260 nm with a Gene Quant II spectrophotometer (Amersham-Pharmacia Biotech,
Orsay, France).
Typing of isolates by enterobacterial repetitive intergenic consensus (ERIC-PCR). To
eliminate replicates within each group of strains, all isolates were typed using ERIC-PCR
with the ERIC-2 primer (5'-AAGTAAGTGACTGGGGTGAGCG-3') as described (35). PCR
products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and
visualized using UV radiation. The patterns obtained were analyzed using Bionumerics
software V1.5 (AppliedMaths, Ghent, Belgium). Single-band differences between isolate
patterns led to the definition of separate genotypes. Only the strains in each group with
different genotypes were included in the analysis.
5
Phylogenetic grouping. Strains were assigned to the E. coli phylogenetic groups using a
triplex PCR based on the presence or absence of three DNA fragments: chuA, yjaA and
TspE4C2, as previously described (4).
Integron detection. For the detection of class 1, 2 and 3 integrons, isolates were tested for the
presence of genes intI1, intI2, and intI3 by a triplex real-time PCR on an ABI Prism 7000
SDS thermocycler (Applied BioSystems, Courtaboeuf, France) using specific primer pairs,
also as previously described (32).
Statistical analysis. Each isolate was regarded as independent sample, since each was derived
from a single host and had a single ERIC-PCR pattern. Rates were compared using the chisquare test or, when samples were small, Fisher’s exact test. A p value of <0.05 was
considered significant.
6
Results
ERIC-PCR patterns. After processing by ERIC-PCR, the 198 isolates studied displayed 173
different patterns. Similar patterns were seen in 2 isolates in the G1 group, 10 in G2 and 13 in
G3. After discarding the duplicates, 64, 56 and 53 isolates from the G1, G2 and G3 groups
respectively were further analyzed.
Phylogenetic group distribution. In all, 77/173 (44.5%) of the strains tested belonged to
phylogenetic group B2, 45/173 (26%) to group A, 17/173 (9.8%) to group B1 and 34/173
(19.7%) to group D. Phylogenetic group distribution in E. coli strain groups G1, 2, and 3 is
shown in Table 1. It differed significantly in G1 and G3 (p<0.05) and also in G2 and G3
(p<0.05) but not in G1 and G2. The prevalence of group B2 was significantly lower in the
most resistant G3 strains than in susceptible G1 strains (12/53, 22.6%, versus 37/64, 57.8%,
chi2, p<0.001) and in G2 strains (12/53, 22.6% versus 28/56, 50%, p<0.01). Interestingly, G3
and G2 only differed by their susceptibility to NAL. The prevalence of phylogenetic group B2
isolates was not significantly different between strains of the G1 and G2 groups, despite their
difference of resistance to AMX and TMP-SMZ (Table 1). The prevalence of phyogenetic
group A was significantly higher in the most resistant G3 strains than in G1 or G2 strains
(21/53, 39.6% versus 13/64, 20.3% or versus 11/56, 19.6%, p<0.01).
Integron prevalence and distribution. In all, an integrase-encoding gene was detected in
99/173 (57.2%) of the strains tested: 80/99 (80.8%) had intI1, 8/99 (8.1%) intI2, and 11/99
(11.1%), both intI1 and intI2. No strain included intI3. The prevalence of integrons was very
high in the resistant G2 and G3 strains (49/56, 87.5% and 50/53, 94.3% respectively). No
integrons were found in the susceptible G1 strains (Table 1). When all strains were analyzed
together, integron prevalence was significantly lower in phylogenetic group B2 strains than in
group A, B1 and D strains (44.2% versus 66.7, 76.5 and 64.7 % respectively, p<0.05 in each
7
instance), but these differences disappeared when samples were grouped according to
resistance patterns (Table 2).
8
Discussion
With a carefully age and sex matched cohort of hospitalized UTI patients, we show, as
reported in various conditions (18,21,23,28,34,36), that the B2 E. coli strains are less resistant
to antibiotics than other phylogenetic E. coli groups, and that this concerns quinolones in
particular. Although the molecular mechanism is still unknown, these data indicate a role of
the genomic background in the emergence of the resistance gained by point mutations, as
reported also for extended beta-lactamases which are mobile elements horizontally transferred
(3).
As expected, we found no integrons in the sensitive strains in the G1 group. On the
other hand, in the resistant G1 and G2 strains, almost all the isolates carried an integron. This
is the higher proportion of integrons reported in the literature (25) probably because all the
strains tested were from hospitalized UTI patients submitted to high antibiotic selective
pressure. In this environment, we do not observed the pattern resistance/absence of integrons
reported for wild animal strains where the antibiotic pressure is very low (33). We statistically
confirm with UTI strains exhibiting a 57 % prevalence of integrons the trend of the
underrepresentation of B2 integron positive strains that has been observed for commensal
strains where the prevalence of integrons is of 15 % (32). However, when considering only
resistant strains, a reflect of strong antibiotic selective pressure, integrons are widespread and
common means enabling bacteria causing infections to adapt to the highly selective
environment, whatever their phylogenetic background.
In conclusion, our results shed further light on the complex interactions between the
environment, represented partly by the antibiotic selective pressure, and the genomic
constraints, represented by the genetic background, in the emergence of the resistance to
antibiotics, revealing a step-by-step strategy. Under very low selective pressure, resistance
emerges without integrons. When the antibiotic pressure raises, quinolone and integron
9
mediated resistance occurs outside phylogenetic group B2. With strong antibiotic selective
pressure, integrons are highly prevalent and widespread whatever the phylogenetic group.
10
Table 1: Distribution of the phylogenetic groups and prevalence of integrons in the populations
of E. coli tested
Resistance groups
Repartition of the phylogenetic groups (%)
A
B1
D
B2
Prevalence of integrons (%)
intI1
intI2
intI
G1(a) (n=64)
20.3
6.3
15.6
57.8
0
0
0
(n=56)
19.7
8.9
21.4
50
83.9
10.7
87.5
G3 (c) (n=53)
39.6
15.1
22.6
22.6
83
24.5
94.3
G2
(b)
All (n=173)
26
9.8
19.7
44.5
52.6
11
57.2
(a)
G1 comprises strains susceptible to amoxicillin (AMX), cotrimoxazol (TMP-SMZ) and
nalidixic acid (NA)
(b)
G2 comprises strains resistant to TMP-SMZ and AMX but susceptible to NA
(c)
G3 comprises strains resistant to TMP-SMZ, AMX and NA
Table 2: Distribution of integrons between the four phylogenetic groups in the three groups with
different antibiotic phenotype studied
Resistance
G1+G2+G3
G1(a) (n=64)
G2(b) (n=56)
G3(c) (n=53)
groups
(n=173)
Phylogenetic
A B1 D B2
A B1 D B2
A B1 D B2
A B1 D B2
groups
Prevalence of
integron (%)
Class 1
0 0 0 0
91 100 92 75
76 87 85 83
58 71 65 40
integron
Class 2
0 0 0 0
0
0 0 21
33 25 8 25
16 12 3 12
integron
Class 1and/or
class 2
0 0 0 0
91 100 92 82
95 100 92 92
67 76 65 44
integron
(a)
G1 comprises strains susceptible to amoxicillin (AMX), cotrimoxazol (TMP-SMZ) and
nalidixic acid (NA)
(b)
G2 comprises strains resistant to TMP-SMZ and AMX but susceptible to NA
(c)
G3 comprises strains resistant to TMP-SMZ, AMX and NA
11
References
1.
Bingen, E., B. Picard, N. Brahimi, S. Mathy, P. Desjardins, J. Elion, and E.
Denamur. 1998. Phylogenetic analysis of Escherichia coli strains causing neonatal
meningitis suggests horizontal gene transfer from a predominant pool of highly
virulent B2 group strains. J. Infect. Dis. 177:642-50.
2.
Boucher, Y., M. Labbate, J. E. Koenig, and H. W. Stokes. 2007. Integrons:
mobilizable platforms that promote genetic diversity in bacteria. Trends. Microbiol.
15:301-9.
3.
Branger, C., O. Zamfir, S. Geoffroy, G. Laurans, G. Arlet, H. V. Thien, S.
Gouriou, B. Picard, and E. Denamur. 2005. Genetic background of Escherichia coli
and extended-spectrum beta-lactamase type. Emerg. Infect. Dis. 11:54-61.
4.
Clermont, O., S. Bonacorsi, and E. Bingen. 2000. Rapid and simple determination
of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66:4555-8.
5.
Correia, M., F. Boavida, F. Grosso, M. J. Salgado, L. M. Lito, J. M. Cristino, S.
Mendo, and A. Duarte. 2003. Molecular characterization of a new class 3 integron in
Klebsiella pneumoniae. Antimicrob. Agents Chemother. 47:2838-43.
6.
Desjardins, P., B.Picard, B. Kaltenbock, J. Elion, and E. Denamur. 1995. Sex in
Escherichia coli does not disrupt the clonal structure of the population: evidence from
random amplified polymorphic DNA and restriction-fragment-length polymorphism.
J. Mol. Evol. 41:440-8.
7.
Escobar-Paramo, P., K. Grenet, A. Le Menac'h, L. Rode, E. Salgado, C. Amorin,
S. Gouriou, B. Picard, M. C. Rahimy, A. Andremont, E. Denamur, and R.
Ruimy. 2004. Large-scale population structure of human commensal Escherichia coli
isolates. Appl. Environ. Microbiol. 70:5698-700.
8.
Escobar-Páramo P, A. Le Menac'h, T. Le Gall, C. Amorin, S. Gouriou, B. Picard,
D. Skurnik, and E. Denamur. 2006. Identification Identification of forces shaping
the commensal Escherichia coli genetic structure by comparing animal and human
isolates. Environ. Microbiol. 8:1975-84.
9.
Fluit, A. C., and F. J. Schmitz. 1999. Class 1 integrons, gene cassettes, mobility, and
epidemiology. Eur. J. Clin. Microbiol. Infect. Dis. 18:761-70.
12
10.
Gordon, D. M., and A. Cowling. 2003. The distribution and genetic structure of
Escherichia coli in Australian vertebrates: host and geographic effects. Microbiology
149:3575-86.
11.
Gordon, D. M., S. E. Stern, and P. J. Collignon. 2005. Influence of the age and sex
of human hosts on the distribution of Escherichia coli ECOR groups and virulence
traits. Microbiology 151:15-23.
12.
Goullet, P., and B. Picard. 1990. Electrophoretic type B2 of carboxylesterase B for
characterisation of highly pathogenic Escherichia coli strains from extra-intestinal
infections. J. Med. Microbiol. 33:11-6.
13.
Hall, R. M., and C. M. Collis. 1995. Mobile gene cassettes and integrons: capture
and spread of genes by site-specific recombination. Mol. Microbiol. 15:593-600.
14.
Herzer, P. J., S. Inouye, M. Inouye, and T. S. Whittam. 1990. Phylogenetic
distribution of branched RNA-linked multicopy single-stranded DNA among natural
isolates of Escherichia coli. J. Bacteriol. 172:6175-81.
15.
Hilali, F., R. Ruimy, P. Saulnier, C. Barnabe, C. Lebouguenec, M. Tibayrenc,
and A. Andremont. 2000. Prevalence of virulence genes and clonality in Escherichia
coli strains that cause bacteremia in cancer patients. Infect. Immun. 68:3983-9.
16.
Johnson, J. R., P. Goullet, B. Picard, S. L. Moseley, P. L. Roberts, and W. E.
Stamm. 1991. Association of carboxylesterase B electrophoretic pattern with presence
and expression of urovirulence factor determinants and antimicrobial resistance among
strains of Escherichia coli that cause urosepsis. Infect. Immun. 59:2311-5.
17.
Johnson, J. R., M. A. Kuskowski, A. Gajewski, S. Soto, J. P. Horcajada, M. T.
Jimenez de Anta, and J. Vila. 2005. Extended virulence genotypes and phylogenetic
background of Escherichia coli isolates from patients with cystitis, pyelonephritis, or
prostatitis. J. Infect. Dis. 191:46-50.
18.
Johnson, J. R., M. A. Kuskowski, K. Owens, A. Gajewski, and P. L. Winokur.
2003. Phylogenetic origin and virulence genotype in relation to resistance to
fluoroquinolones and/or extended-spectrum cephalosporins and cephamycins among
Escherichia coli isolates from animals and humans. J. Infect. Dis. 188:759-68.
19.
Johnson, J. R., T. T. O'Bryan, P. Delavari, M. Kuskowski, A. Stapleton, U.
Carlino, and T. A. Russo. 2001. Clonal relationships and extended virulence
genotypes among Escherichia coli isolates from women with a first or recurrent
episode of cystitis. J. Infect. Dis. 183:1508-17.
13
20.
Johnson, J. R., E. Oswald, T. T. O'Bryan, M. A. Kuskowski, and L. Spanjaard.
2002. Phylogenetic distribution of virulence-associated genes among Escherichia coli
isolates associated with neonatal bacterial meningitis in the Netherlands. J. Infect. Dis.
185:774-84.
21.
Johnson, J. R., C. van der Schee, M. A. Kuskowski, W. Goessens, and A. van
Belkum. 2002. Phylogenetic background and virulence profiles of fluoroquinoloneresistant clinical Escherichia coli isolates from the Netherlands. J. Infect. Dis.
186:1852-6.
22.
Kaper, J. B., J. P. Nataro, and H. L. Mobley. 2004. Pathogenic Escherichia coli.
Nat. Rev. Microbiol. 2:123-40.
23.
Kuntaman, K., E. S. Lestari, J. A. Severin, I. M. Kershof, N. M. Mertaniasih, M.
Purwanta, U. Hadi, J. R. Johnson, A. van Belkum, and H. A. Verbrugh. 2005.
Fluoroquinolone-resistant Escherichia coli, Indonesia. Emerg. Infect. Dis. 11:1363-9.
24.
Lecointre, G., L. Rachdi, P. Darlu, and E. Denamur. 1998. Escherichia coli
molecular phylogeny using the incongruence length difference test. Mol. Biol. Evol.
15:1685-95.
25.
Leverstein-van Hall, M. A., M. B. HE, T. D. AR, A. Paauw, A. C. Fluit, and J.
Verhoef. 2003. Multidrug resistance among Enterobacteriaceae is strongly associated
with the presence of integrons and is independent of species or isolate origin. J. Infect.
Dis. 187:251-9.
26.
Leverstein-Van Hall, M. A., A. Paauw, A. T. Box, H. E. Blok, J. Verhoef, and A.
C. Fluit. 2002. Presence of integron-associated resistance in the community is
widespread and contributes to multidrug resistance in the hospital. J. Clin. Microbiol.
40:3038-40.
27.
Mazel, D. 2006. Integrons: agents of bacterial evolution. Nat. Rev. Microbiol. 4:60820.
28.
Moreno, E., G. Prats, M. Sabate, T. Perez, J. R. Johnson, and A. Andreu. 2006.
Quinolone, fluoroquinolone and trimethoprim/sulfamethoxazole resistance in relation
to virulence determinants and phylogenetic background among uropathogenic
Escherichia coli. J. Antimicrob. Chemother. 57:204-11.
29.
Picard, B., and P. Goullet. 1989. Correlation between electrophoretic types B1 and
B2 of carboxylesterase B and sex of patients in Escherichia coli urinary tract
infections. Epidemiol. Infect. 103:97-103.
14
30.
Picard, B., J. S. Garcia, S. Gouriou, P. Duriez, N. Brahimi, E. Bingen, J. Elion,
and E. Denamur. 1999. The link between phylogeny and virulence in Escherichia
coli extraintestinal infection. Infect. Immun. 67:546-53
31.
Selander, R. K. and Levin, B. R. 1980. Genetic diversity and structure in Escherichia
coli populations. Science 210:545-7.
32.
Skurnik, D., A. Le Menac'h, D. Zurakowski, D. Mazel, P. Courvalin, E.
Denamur, A. Andremont, and R. Ruimy. 2005. Integron-associated antibiotic
resistance and phylogenetic grouping of Escherichia coli isolates from healthy
subjects free of recent antibiotic exposure. Antimicrob. Agents Chemother. 49:3062-5.
33.
Skurnik, D., R. Ruimy, A. Andremont, C. Amorin, P. Rouquet, B. Picard, and E.
Denamur. 2006. Effect of human vicinity on antimicrobial resistance and integrons in
animal faecal Escherichia coli. J. Antimicrob. Chemother. 57:1215-9.
34.
Velasco, M., J. P. Horcajada, J. Mensa, A. Moreno-Martinez, J. Vila, J. A.
Martinez, J. Ruiz, M. Barranco, G. Roig, and E. Soriano. 2001. Decreased
invasive capacity of quinolone-resistant Escherichia coli in patients with urinary tract
infections. Clin. Infect. Dis. 33:1682-6.
35.
Versalovic, J., T. Koeuth, and J. R. Lupski. 1991. Distribution of repetitive DNA
sequences in eubacteria and application to fingerprinting of bacterial genomes.
Nucleic. Acids Res. 19:6823-31.
36.
Vila, J., K. Simon, J. Ruiz, J. P. Horcajada, M. Velasco, M. Barranco, A. Moreno,
and J. Mensa. 2002. Are quinolone-resistant uropathogenic Escherichia coli less
virulent? J. Infect. Dis. 186:1039-42.
37.
Wiener, J., J. P. Quinn, P. A. Bradford, R. V. Goering, C. Nathan, K. Bush, and
R. A. Weinstein. 1999. Multiple antibiotic-resistant Klebsiella and Escherichia coli in
nursing homes. JAMA 281:517-23
15