INSERM U4581, Laboratoire détudes de génétique bactérienne dans les infections de lenfant (EA3105)2, Hôpital Robert Debré, 48 Boulevard Sérurier 75019 Paris, France
Département dAnthropologie Génétique, Bordeaux II, 3 Place de la Victoire, 33000 Bordeaux, France3
Laboratoire de Microbiologie, Faculté de médecine de Brest, 22 Avenue Camille Desmoulins, B.P. 815, 29285 Brest Cedex, France4
Author for correspondence: Erick Denamur. Tel: +33 1 400 31916. Fax: +33 1 400 31903. e-mail: denamur{at}infobiogen.fr
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ABSTRACT |
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Keywords: Escherichia coli, phylogeny, commensal, virulence
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INTRODUCTION |
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The population structure of several pathogenic isolates has been extensively studied but little is known about the structure of commensal strain populations. It is therefore mandatory to determine the intraspecies phylogenetic relationships of E. coli isolates from the normal gut flora of healthy humans, to establish a background database for further studies on pathogenic strains. In this study, we used a recently described method facilitating the rapid and simple determination of E. coli phylogenetic group (Clermont et al., 2000 ) in 168 non-epidemiologically related isolates from three geographically distinct human populations. This technique is based on a triplex PCR using a combination of two genes (chuA and yjaA) and an anonymous DNA fragment. The results obtained were strongly correlated with those obtained by multilocus enzyme electrophoresis and ribotyping methods (Clermont et al., 2000
). The distributions of several known extraintestinal virulence factors (pap, afa, sfa/foc adhesin-encoding operons, hly and aer operons and ibeA and cnf1genes) were also determined.
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METHODS |
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PCR grouping.
The phylogenetic group to which the E. coli strains belonged was determined by a PCR-based method as described previously (Clermont et al., 2000 ). Briefly, a two-step triplex PCR was performed directly on 3 µl bacterial lysate. The primer pairs used were ChuA.1 (5'-GACGAACCAACGGTCAGGAT-3')/ChuA.2 (5'-TGCCGCCAGTACCAAAGACA-3'), YjaA.1 (5'-TGAAGTGTCAGGAGACGCTG-3')/YjaA.2 (5'-ATGGAGAATGCGTTCCTCAAC-3') and TspE4C2.1 (5'-GAGTAATGTCGGGGCATTCA-3')/TspE4C2.2 (5'-CGCGCCAACAAAGTATTACG-3'). The PCR steps were as follows: denaturation for 4 min at 94 °C, 30 cycles of 5 s at 94 °C and 10 s at 59 °C, and a final extension step of 5 min at 72 °C. The data of the three amplifications resulted in assignment of the strains to phylogenetic groups as follows: chuA+, yjaA+, group B2; chuA+, yjaA-, group D; chuA-, TspE4.C2+, groupB1; chuA-, TspE4.C2-, group A (Clermont et al., 2000
).
Detection of virulence factors.
Virulence factors were detected by amplifying the corresponding gene (ibeA, pap, afa, sfa/foc, hly, cnf1 and aer) from DNA by PCR as described previously (Picard et al., 1999 , 2001
).
Statistical analysis.
In addition to the data obtained experimentally in this work, the commensal strains were compared with two previously analysed collections of extraintestinal pathogenic strains (Bingen et al., 1998 ; Picard et al., 1999
). The frequencies of various phylogenetic groups in the human populations were compared using the
2 test and the distributions of the number of virulence factors were compared using the MannWhitney U-test. All these tests were carried out using the Statview 4.5 software (Abacus Concept). A P value less than 0·05 was considered to show a significant difference.
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RESULTS AND DISCUSSION |
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Distribution of extraintestinal virulence genes among human commensal strains
Extraintestinal virulence genes were rarely detected among human commensal strains, as none of the seven virulence genes studied was detected in 74·4% of strains. Only 11·3, 1·2 and 3·6% of strains contained the pap, afa and sfa/foc adhesin-coding operons, respectively. The aer determinant was found in 16% of strains whereas the remaining virulence determinants were detected in less than 5% of strains (Table 2). These proportions differed according to the phylogenetic origin of the strains. The strains most frequently bearing virulence factors were those belonging to phylogenetic group B2, in which two or three virulence genes were often detected per strain (Table 3
). The 43 strains harbouring at least one virulence gene (25·6% of the 168 strains) included 28 harbouring one, 9 harbouring two, 2 harbouring three, 2 harbouring four and 2 harbouring five virulence genes (Table 3
). The strains from Mali harboured fewer virulence factors than did the Croatian (U-test, P=0·005) and French (U-test, P=0·0039) isolates, as only one Malian strain (B2 group) possessed two virulence factors versus 23 strains in Croatia and 19 strains in France.
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Comparison of human commensal strain data with data obtained from two extraintestinal pathogen collections
The phylogenetic grouping of strains used for comparison were all obtained by the PCR triplex method. The 69 neonatal meningitis strains (Bingen et al., 1998 ) and the 49 strains isolated from miscellaneous extraintestinal infections in adults (Picard et al., 1999
) were reanalysed by the PCR grouping method and the results obtained were strongly correlated with those of ribotyping (Clermont et al., 2000
and data not shown). Virulence determinants were also detected using the same approach. The cnf1 data for the pathogen collections are unpublished.
Of the 118 extraintestinal pathogenic strains, 110 (93%) harboured at least one virulence gene. The human commensal strains had fewer virulence factors than the extraintestinal pathogenic strains (U-test, P<0·0001). This was also true for the frequencies of each individual virulence determinant, except for afa, which was under-represented (Table 2).
Pathogenic strains were distributed among phylogenetic groups as follows: A, 11 strains (9%); B1, 3 strains (2·5%); D, 19 strains (16%) and B2, 85 strains (72%). If virulence determinant frequencies were compared by phylogenetic group between the commensal and extraintestinal pathogenic series, the commensal strains of phylogenetic groups A, B1 and D appeared to possess fewer virulence determinants (U-test, P<0·0001,=0·0017 and <0·0001, respectively). In contrast, no significant difference was observed between the phylogenetic B2 strains. This absence of difference was also found for each virulence gene (Table 3).
In addition, confirming that the strains of phylogenetic group B2 are more frequent among extraintestinal pathogenic strains (4072%) than among commensal strains (911%) (Goullet & Picard, 1986b ; Johnson et al., 1991
; Bingen et al., 1998
; Picard et al., 1999
; Hilali et al., 2000
; this work), our homogeneous dataset also increases our understanding of the emergence of virulence in human extraintestinal infections. The smaller number of virulence determinants in commensal strains of phylogenetic groups A, B1 and D than in the corresponding pathogenic strains is consistent with the hypothesis (Bingen et al., 1998
; Lecointre et al., 1998
; Picard et al., 1999
) that these strains acquire virulence factors by horizontal transfer, enabling them to become virulent and to invade immunocompetent hosts (Picard & Goullet, 1988
) from the hosts intestinal reservoir. In contrast, the strains of phylogenetic group B2 in the commensal flora appear to be potentially virulent. This intrinsic virulence may explain why strains of phylogenetic group B2 are frequently isolated from patients with extraintestinal infections, despite their rarity in the gut flora.
Concluding remarks
This work on a relatively small number of commensal strains clearly demonstrates that the commensal flora has a complex population structure, which depends on ecotype. As commensal enteric isolates are believed to be the reservoirs of pathogenic strains, it will be interesting to determine whether the geographic differences between E. coli commensal populations are associated with differences in the distribution of diseases caused by different types of E. coli. Studies on a large number of commensal strains including precise geographic, socio-economic and medical data are needed to provide further insight into the emergence of virulent E. coli clones.
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ACKNOWLEDGEMENTS |
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Received 25 October 2000;
revised 1 February 2001;
accepted 14 February 2001.