Department of Obstetrics and Gynaecology, Institute of Clinical Medicine1 and Department of Infection Biology, Institute of Basic Medical Sciences2, University of Tsukuba, Tsukuba, 305-8575, Japan
Osaka Prefectural Institute of Public Health, OSaka, 537-0025, Japan2
Author for correspondence: William Ba-Thein. Tel: +81 298 53 3354. Fax: +81 298 53 3354. e-mail: bathein{at}md.tsukuba.ac.jp
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ABSTRACT |
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Keywords: vagina, Escherichia coli, extraintestinal virulence factors, phylogenetic grouping, O:K:H antigens
Abbreviations: CSF, cerebrospinal fluid; HNM, H non-motile; MLEE, multilocus enzyme electrophoresis; NMEC, neonatal meningitis E. coli; PAI, pathogenicity-associated island marker; UPEC, uropathogenic E. coli; VEC, vaginal E. coli; VF, virulence factor
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INTRODUCTION |
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Vaginal colonization with E. coli is associated with various genitourinary, obstetric and neonatal complications, such as the severe form of pelvic inflammatory disease (Heinonen & Miettinen, 1994 ; Larsen & Galask, 1980
), urinary tract infections (OGrady et al., 1970
; Stamey & Sexton, 1975
), very-low-birth-weight infants (Krohn et al., 1997
) and early-onset neonatal septicaemia and meningitis (Sarff et al., 1975
; Schiffer et al., 1976
). Vaginal E. coli has also been reported to be sexually transmissible to a male partner (Hebelka et al., 1993
).
The E. coli implicated in various human infections can be broadly classified as intestinal and extraintestinal. Among the extraintestinal E. coli, uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC) have been characterized in some detail by diverse approaches including phylogenetic analysis, serotyping and molecular typing such as virulence factor (VF) profiling, multilocus enzyme electrophoresis (MLEE), outer-membrane protein profiling and plasmid profiling. Vagina-colonizing E. coli, however, remains largely uncharacterized with regard to its genotypes, serotypes, clonality and phylogenetic linkage with other E. coli.
In this paper we report on the characteristics of E. coli isolates from vaginal swab samples of both pregnant and non-pregnant women as determined by extraintestinal E. coli VF profiling, PCR-based phylogenetic grouping and O:H serotyping. We also report on the results of comparative analyses with E. coli isolates from normal stool samples and with previous data from stool, urine, blood and cerebrospinal fluid (CSF) isolates (Bingen et al., 1997 , 1998
; Duriez et al., 2001
; Johnson & Stell, 2000
; Johnson et al., 2002
; Miyazaki et al., 2002
; Sarff et al., 1975
; Siitonen, 1992
; Tsukamoto, 1997
; Yamamoto et al., 1995b
).
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METHODS |
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For comparative analyses with respect to the characteristics of VEC, previously published data on the following were included: (i) VF profiles of 2245 E. coli strains of various sources from East Japan (Ibaraki), West Japan (Kyoto, Osaka, Shiga) and other countries, including strains isolated from adult stool [n=80 (Yamamoto et al., 1995a ), n=287 (Siitonen, 1992
)], neonatal stool [n=1169 (Sarff et al., 1975
)], neonatal CSF (bacterial meningitis) [n=67 (Bingen et al., 1997
), n=70 (Johnson et al., 2002
)], neonatal blood (septicaemia) [n=47 (Bingen et al., 1997
)], adult urine (acute simple cystitis) [n=194 (Yamamoto et al., 1995b
), n=256 (Miyazaki et al., 2002
)] and adult blood (urosepsis) [n=75 (Johnson & Stell, 2000
)]; (ii) phylogenetic analysis of 307 E. coli strains, including strains from normal stool [n=168 (Duriez et al., 2001
)], neonatal CSF (bacterial meningitis) [n=69 (Bingen et al., 1998
), n=70 (Johnson et al., 2002
)]; (iii) profiles of O:H serotypes and K1 antigen of 320 extraintestinal E. coli strains from neonatal CSF (bacterial meningitis) (n=52), neonatal blood (septicaemia) (n=74) and adult urine (acute simple cystitis) (n=194) from our previous study (Tsukamoto, 1997
).
Sample collection and processing.
After obtaining approval from the Ethical Committees of the hospitals concerned and informed consent from the patients, we collected clinical samples. The vaginal swabs were taken from the posterior vaginal fornix by experienced interns and were immediately streaked onto Trypticase soy/5% sheep blood agar and Drigalsky agar for aerobic cultures. The same swabs were used for microbial identification by Gram staining. Isolates that had been preliminarily identified as E. coli by colony morphology and Gram-stain reactions were confirmed using an API 20E (bioMérieux) kit. The normal stool E. coli strains isolated from Drigalsky agar were also confirmed using an API 20E (bioMérieux) kit. Boiled lysates from at least two batches of overnight-grown bacterial cultures were used as PCR templates for the determination of VFs and phylogenetic groups.
VF profiling.
The prevalence of extraintestinal VFs among E. coli isolates was examined by a multiplex PCR-based screening method that uses the primer pairs described elsewhere: papC (pilus associated with pyelonephritis) [pap1/2: gacggctgtactgcagggtgtggcg/atatcctttctgcagggatgcaata (Le Bouguenec et al., 1992 )]; sfaDE (S fimbriae) [sfa1/2: ctccggagaactgggtgcatcttac/cggaggagtaattacaaacctggca (Johnson & Brown, 1996
); afa/draBC (afimbrial adhesins) [afa1/2: gctgggcagcaaactgataactctc/catcaagctgtttgttcgtccgccg (Le Bouguenec et al., 1992
)]; iucD (aerobactin synthesis) [aer1/2: taccggattgtcatatgcagaccgt/aatatcttcctccagtccggagaag (Yamamoto et al., 1995a
)]; cnf1 (cytotoxic necrotizing factor) [cnf1/2: aagatggagtttcctatgcaggag/cattcagagtcctgccctcattatt (Yamamoto et al., 1995a
); hlyA (haemolysin) [hly1/2: aacaaggataagcactgttctggct/accatataagcggtcattcccgtca (Yamamoto et al., 1995a
); PAI (pathogenicity-associated island marker) [RPAi f/r: ggacatcctgttacagcgcgca/tcgccaccaatcacagccgaac (Johnson & Stell, 2000
)]; fimH (mannose-specific adhesin subunit of type 1 fimbriae) [fimH f/r: tgcagaacggataagccgtgg/gcagtcacctgccctccggta (Johnson & Stell, 2000
)]; and ibeA (invasion brain endothelium) [ibe10 f/r: aggcaggtgtgcgccgcgtac (Huang et al., 1995
) and tggtgctccggcaaaccatgc (Johnson & Stell, 2000
)].
A PCR mixture (50 µl) containing 5 µl bacterial lysate, 62·5 µM dNTPs, 5 µl 10x PCR buffer (100 mM Tris/HCl, pH 8·8, 500 mM KCl, 15 mM MgCl2, 1% Triton X-100), Ex Taq DNA polymerase (0·03 U µl-1) (TaKaRa Shuzo) and primer set A (20 pmol each of pap1/2, sfa1/2, afa1/2, aer1/2 and cnf1/2 primers; 30 pmol each of hly1/2 primers) or primer set B (10 pmol each of RPAi f/r, fimH f/r and ibe10 f/r primers) was amplified in a thermal cycler (PTC-100; MJ Research) for 30 cycles with a profile of 94 °C for 1 min, 63 °C for 30 s and 72 °C for 3 min, followed by a final extension at 72 °C for 3 min. Amplified products were separated in 2% agarose gels, and ethidium bromide-stained gels were visualized with an ultraviolet transilluminator for photographic imaging.
Three methods were used for K1 antigen determination: (i) PCR-based detection of neuS (K1-specific gene) (Tsukamoto, 1997 ), (ii) antiserum and (iii) bacteriophage-based detection using five K1-specific bacteriophages (Gross et al., 1977
). Five bacteriophages specific to the K1 polysaccharide antigen of E. coli were kindly supplied by Dr B. Rowe (Central Public Health Laboratory, London, UK) (Gross et al., 1977
).
Phylogenetic analysis.
Phylogenetic classification of E. coli isolates was determined using triplex PCR-based phylotyping described by Clermont et al. (2000) , which is a simple method comparable to MLEE-based typing. Briefly, genomic DNA of bacterial strains was amplified by triplex PCR using primers targeted to three markers, chuA, yjaA and TspE4.C2. The phylogenetic grouping was made on the basis of the presence of specific PCR-amplified fragments as follows: group B2 (chuA+, yjaA+, TspE.C2±), group D (chuA+, yjaA-, TspE.C2±), group B1 (chuA-, yjaA±, TspEC2+) and group A (chuA-, yjaA±, TspE.C2-).
O:H serotyping.
The E. coli isolates were serotyped in the Osaka Prefectural Institute of Public Health, Japan, following the method described by Orskov & Orskov (1975) . The O-antigen confirmation for some questionable isolates was performed by the Statens Serum Institute, Denmark. PCR-based H7 typing (Tsukamoto & Kanki, 1999
) was used to assign H7 to some non-motile (HNM) strains.
Statistical analysis.
Significance of differences between the variables was tested with the 2 test and the Fishers exact test.
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RESULTS |
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Phylogenetic groups
There were no significant differences in the distribution of phylogenetic groups between the VEC subsets (non-pregnant vs pregnant or asymptomatic vs symptomatic) (Table 4).
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Among the 31 VEC serotypes, 10 serotypes (O1:K1:H7, O2:K1:H7, O4:H5, O6:H1, O6:NM, O16:K1:H6, O16:K1:NM, O18ac:K1:H7, O75:HNM and O77:HNM) were also identified in the strains from urine and neonates. Seven ONT:K1:H4 strains were observed among the VEC isolates. All of them belonged to phylogenetic group B2 and had identical VF profiles; they all were positive for ibeA, PAI and fimH, and negative for other VFs.
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DISCUSSION |
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Taken together, the VEC strains are very similar to other extraintestinal E. coli with respect to the overall distribution of VFs. With their common VF profiles, all extraintestinal E. coli can be considered virulent clones (Finlay & Falkow, 1997 ; Ochman et al., 2000
). This in turn supports the concept of the recently proposed term, extraintestinal pathogenic E. coli (ExPEC) (Russo & Johnson, 2000
).
Among four recognized phylogenetic groups (A, B1, B2 and D) (Herzer et al., 1990 ; Selander et al., 1996
), the virulent extraintestinal E. coli strains belong mainly to group B2 and, to a lesser extent, to group D, whereas most commensal strains belong to group A (Bingen et al., 1998
; Johnson & Stell, 2000
; Picard et al., 1999
). The fact that most of the VEC strains (92%) were grouped into B2 or D suggests that most of the VEC strains are potentially virulent. It should be noted, however, that this high occurrence of group B2 (76%) in the VEC strains might have originated from the higher occurrence of group B2 (44%) in the commensal faecal E. coli strains of Japanese populations. Duriez et al. (2001)
has suggested that the commensal strains are phylogenetically distributed among geographically distinct human populations. This hypothesis could explain the difference between the distribution of phylogenetic groups of Japanese faecal strains (this study) and that of European and African faecal strains (Duriez et al., 2001
). Therefore, it is worth examining more VEC isolates from distinct geographical populations to better understand the role of VEC in extraintestinal infections.
The serogroups O1, O2, O4, O6, O18, O25 and O75 found in three or more VEC strains have also been reported as the common serogroups in UPEC (Czirok et al., 1986 ; Korhonen et al., 1985
; Mulder et al., 1984
; Orskov et al., 1982
; Sandberg et al., 1988
; Stenqvist et al., 1987
; Vaisanen-Rhen et al., 1984
) and E. coli of neonatal meningitis or septicaemia (Cross et al., 1984
; Korhonen et al., 1985
; McCabe et al., 1978
; McCracken et al., 1974
; Robbins et al., 1974
). Among 10 serotypes that were distributed elsewhere in the VEC and other extraintestinal strains of our study, O18ac:K1:H7 strains of cystitis, neonatal bacterial meningitis and faecal origin have already been shown to be clonally derived (Johnson et al., 2001
). The strains belonging to the remaining nine serotypes could also be clonal, though further examination is needed for confirmation.
Inasmuch as 39% (12/31) of serotypes in the VEC strains were restricted to the vagina and the remaining 61% (19/31) can be found elsewhere, it is likely that a subset of the VEC, the former group, is confined locally as the vagina-specific residents whilst the latter group can spread to and exist in other extraintestinal sites.
In conclusion, our data demonstrate that the VEC share common VF profiles, phylogenetic groups and serotypes with E. coli strains from urinary and neonatal (blood and CSF) origins. This study provides additional evidence for a link among extraintestinal E. coli, which in turn supports the concept that VEC are a reservoir along the faecalvaginalurinary/neonatal course of transmission in extraintestinal E. coli infections.
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ACKNOWLEDGEMENTS |
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Received 20 March 2002;
revised 29 May 2002;
accepted 30 May 2002.
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