# The Plasmid of Escherichia coli Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic E. coli Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model

Chantal Peigne, Philippe Bidet, Farah Mahjoub-Messai, Céline Plainvert, Valérie Barbe, Claudine Médigue, Eric Frapy, Xavier Nassif, Erick Denamur, Edouard Bingen, et al.

► To cite this version:

Chantal Peigne, Philippe Bidet, Farah Mahjoub-Messai, Céline Plainvert, Valérie Barbe, et al. The Plasmid of Escherichia coli Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic E. coli Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model. Infection and Immunity, 2009, 77 (6), pp.2272 - 2284. 10.1128/iai.01333-08 . hal-04581643

HAL Id: hal-04581643

https://hal.science/hal-04581643

Submitted on 21 May 2024

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# The Plasmid of Escherichia coli Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic E. coli Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model

Chantal Peigne, Philippe Bidet, Farah Mahjoub-Messai, Céline Plainvert, Valérie Barbe, Claudine Médigue, Eric Frapy, Xavier Nassif, Erick Denamur, Edouard Bingen, et al.

<figure><img src="image_1.png" /></figure>

► To cite this version:

Chantal Peigne, Philippe Bidet, Farah Mahjoub-Messai, Céline Plainvert, Valérie Barbe, et al. The Plasmid of Escherichia coli Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic E. coli Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model. Infection and Immunity, 2009, 77 (6), pp.2272 - 2284. 10.1128/iai.01333-08 . hal-04581643

HAL Id: hal-04581643

https://hal.science/hal-04581643v1

Submitted on 21 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
# The Plasmid of *Escherichia coli* Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic *E. coli* Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model<sup>¶</sup>

Chantal Peigne,¹ Philippe Bidet,¹ Farah Mahjoub-Messai,¹ Céline Plainvert,¹ Valérie Barbe,²

Claudine Médigue,³ Eric Frapy,⁴ Xavier Nassif,⁴ Erick Denamur,⁵

Edouard Bingen,¹ and Stéphane Bonacorsi¹*

*Laboratoire d'Études de Génétique Bactérienne dans les Infections de l'Enfant (EA 3105), Université Paris Diderot, Hôpital Robert Debré, 75019 Paris,¹ Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Génomique, Génoscope, 91057 Evry Cedex,² Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Génomique, Atelier de Génomique Comparative, CNRS UMR8030, 91057 Evry Cedex,³ Institut National de la Santé et de la Recherche Médicale U570 and Université Paris Descartes, 75015 Paris,⁴ and Institut National de la Santé et de la Recherche Médicale U722, 75018 Paris, and Université Paris Diderot, Site Xavier Bichat, 75018 Paris,⁵ France*

Received 31 October 2008/Returned for modification 12 January 2009/Accepted 15 March 2009

A new *Escherichia coli* virulent clonal group, O45:K1, belonging to the highly virulent subgroup B2₁ was recently identified in France, where it accounts for one-third of *E. coli* neonatal meningitis cases. Here we describe the sequence, epidemiology and function of the large plasmid harbored by strain S88, which is representative of the O45:K1 clonal group. Plasmid pS88 is 133,853 bp long and contains 144 protein-coding genes. It harbors three different iron uptake systems (aerobactin, salmochelin, and the *sitABCD* genes) and other putative virulence genes (*iss*, *etsABC*, *ompT*, and *hlyF*). The pS88 sequence is composed of several gene blocks homologous to avian pathogenic *E. coli* plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM. PCR amplification of 11 open reading frames scattered throughout the plasmid was used to investigate the distribution of pS88 and showed that a pS88-like plasmid is present in other meningitis clonal groups such as O18:K1, O1:K1, and O83:K1. A pS88-like plasmid was also found in avian pathogenic strains and human urosepsis strains belonging to subgroup B2₁. A variant of S88 cured of its plasmid displayed a marked loss of virulence relative to the wild-type strain in a neonatal rat model, with bacteremia more than 2 log CFU/ml lower. The salmochelin siderophore, a known meningovirulence factor, could not alone explain the plasmid's contribution to virulence, as a salmochelin mutant displayed only a minor fall in bacteremia (0.9 log CFU/ml). Thus, pS88 is a major virulence determinant related to avian pathogenic plasmids that has spread not only through meningitis clonal groups but also human urosepsis and avian pathogenic strains.

*Escherichia coli* is the second cause of neonatal bacterial meningitis in industrialized countries, after group B streptococci. Despite advances in neonatal care, *E. coli* neonatal meningitis (ECNM) remains a potentially devastating illness, with a mortality rate of 10 to 15% and neurological sequelae in 30 to 50% of cases (8, 20, 28, 29, 62). Like most extraintestinal pathogenic *E. coli* (ExPEC) strains, ECNM strains mainly belong to phylogenetic group B2 and compose a small number of serotypes (O18:K1:H7, O1:K1, O83:K1, and O7:K1) (2, 11, 12, 33, 41, 49). Virulence genes in ECNM strains are involved in two major pathological steps, namely, intravascular multiplication (leading to high-level bacteremia) and blood-brain barrier penetration (38). A new virulent clone, O45:K1:H7, was recently identified in France, where it now accounts for one-third of ECNM isolates (13). The K1 capsular antigen and the salmochelin siderophore are the only two specific virulence traits

known to be shared by this clone and the archetypal strains of clonal group O18:K1:H7, even though the two clonal groups belong to the same highly virulent subgroup B2₁, defined by a specific sequence type (ST) designated ST29<sub>Whittam</sub> (www.shigatox.net), ST95<sub>Achtmann</sub> (www.mlst.net), or B2-IX (in Denamur's scheme) (10, 40). Hence, this new clone may serve to identify new virulence determinants and other genetic factors involved in the pathogenesis of ECNM. Strain S88, representative of clone O45:K1:H7, has been sequenced as part of a whole-genome sequencing project (ColiScope [www.genoscope.cns.fr]) at the Evry Genoscope in France. This strain harbors a large plasmid, designated pS88, that encodes the salmochelin and aerobactin siderophores and may be involved in the virulence of S88 (13).

Here we describe the complete DNA sequence, epidemiology, and function of pS88.

## MATERIALS AND METHODS

**Bacteria.** Strain S88, a representative of the French clonal group (O45:K1:H7) which accounts for one-third of ECNM isolates in this country, was isolated in 1989 from the cerebrospinal fluid of a newborn (Robert Debré Hospital, Paris, France). This strain is known to harbor a large plasmid (>100 kb) encoding at

<footnote>* Corresponding author. Mailing address: Service de Microbiologie, Hôpital Robert-Debré, 48 blvd. Sérurier, 75395 Paris cedex 19, France. Phone: 33 1 40 03 23 40. Fax: 33 1 40 03 24 50. E-mail: stephane.bonacorsi@rdb.aphp.fr.</footnote>

<footnote>† Published ahead of print on 23 March 2009.</footnote>
# The Plasmid of *Escherichia coli* Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic *E. coli* Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model<sup>¶</sup>

Chantal Peigne,¹ Philippe Bidet,¹ Farah Mahjoub-Messai,¹ Céline Plainvert,¹ Valérie Barbe,²

Claudine Médigue,³ Eric Frapy,⁴ Xavier Nassif,⁴ Erick Denamur,⁵

Edouard Bingen,¹ and Stéphane Bonacorsi¹*

*Laboratoire d'Études de Génétique Bactérienne dans les Infections de l'Enfant (EA 3105), Université Paris Diderot, Hôpital Robert Debré, 75019 Paris,¹ Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Génomique, Génoscope, 91057 Evry Cedex,² Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Institut de Génomique, Atelier de Génomique Comparative, CNRS UMR8030, 91057 Evry Cedex,³ Institut National de la Santé et de la Recherche Médicale U570 and Université Paris Descartes, 75015 Paris,⁴ and Institut National de la Santé et de la Recherche Médicale U722, 75018 Paris, and Université Paris Diderot, Site Xavier Bichat, 75018 Paris,⁵ France*

Received 31 October 2008/Returned for modification 12 January 2009/Accepted 15 March 2009

A new *Escherichia coli* virulent clonal group, O45:K1, belonging to the highly virulent subgroup B2₁ was recently identified in France, where it accounts for one-third of *E. coli* neonatal meningitis cases. Here we describe the sequence, epidemiology and function of the large plasmid harbored by strain S88, which is representative of the O45:K1 clonal group. Plasmid pS88 is 133,853 bp long and contains 144 protein-coding genes. It harbors three different iron uptake systems (aerobactin, salmochelin, and the *sitABCD* genes) and other putative virulence genes (*iss*, *etsABC*, *ompT*, and *hlyF*). The pS88 sequence is composed of several gene blocks homologous to avian pathogenic *E. coli* plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM. PCR amplification of 11 open reading frames scattered throughout the plasmid was used to investigate the distribution of pS88 and showed that a pS88-like plasmid is present in other meningitis clonal groups such as O18:K1, O1:K1, and O83:K1. A pS88-like plasmid was also found in avian pathogenic strains and human urosepsis strains belonging to subgroup B2₁. A variant of S88 cured of its plasmid displayed a marked loss of virulence relative to the wild-type strain in a neonatal rat model, with bacteremia more than 2 log CFU/ml lower. The salmochelin siderophore, a known meningovirulence factor, could not alone explain the plasmid's contribution to virulence, as a salmochelin mutant displayed only a minor fall in bacteremia (0.9 log CFU/ml). Thus, pS88 is a major virulence determinant related to avian pathogenic plasmids that has spread not only through meningitis clonal groups but also human urosepsis and avian pathogenic strains.

*Escherichia coli* is the second cause of neonatal bacterial meningitis in industrialized countries, after group B streptococci. Despite advances in neonatal care, *E. coli* neonatal meningitis (ECNM) remains a potentially devastating illness, with a mortality rate of 10 to 15% and neurological sequelae in 30 to 50% of cases (8, 20, 28, 29, 62). Like most extraintestinal pathogenic *E. coli* (ExPEC) strains, ECNM strains mainly belong to phylogenetic group B2 and compose a small number of serotypes (O18:K1:H7, O1:K1, O83:K1, and O7:K1) (2, 11, 12, 33, 41, 49). Virulence genes in ECNM strains are involved in two major pathological steps, namely, intravascular multiplication (leading to high-level bacteremia) and blood-brain barrier penetration (38). A new virulent clone, O45:K1:H7, was recently identified in France, where it now accounts for one-third of ECNM isolates (13). The K1 capsular antigen and the salmochelin siderophore are the only two specific virulence traits

known to be shared by this clone and the archetypal strains of clonal group O18:K1:H7, even though the two clonal groups belong to the same highly virulent subgroup B2₁, defined by a specific sequence type (ST) designated ST29<sub>Whittam</sub> (www.shigatox.net), ST95<sub>Achtmann</sub> (www.mlst.net), or B2-IX (in Denamur's scheme) (10, 40). Hence, this new clone may serve to identify new virulence determinants and other genetic factors involved in the pathogenesis of ECNM. Strain S88, representative of clone O45:K1:H7, has been sequenced as part of a whole-genome sequencing project (ColiScope [www.genoscope.cns.fr]) at the Evry Genoscope in France. This strain harbors a large plasmid, designated pS88, that encodes the salmochelin and aerobactin siderophores and may be involved in the virulence of S88 (13).

Here we describe the complete DNA sequence, epidemiology, and function of pS88.

## MATERIALS AND METHODS

**Bacteria.** Strain S88, a representative of the French clonal group (O45:K1:H7) which accounts for one-third of ECNM isolates in this country, was isolated in 1989 from the cerebrospinal fluid of a newborn (Robert Debré Hospital, Paris, France). This strain is known to harbor a large plasmid (>100 kb) encoding at

<footnote>* Corresponding author. Mailing address: Service de Microbiologie, Hôpital Robert-Debré, 48 blvd. Sérurier, 75395 Paris cedex 19, France. Phone: 33 1 40 03 23 40. Fax: 33 1 40 03 24 50. E-mail: stephane.bonacorsi@rdb.aphp.fr.</footnote>

<footnote>¶ Published ahead of print on 23 March 2009.</footnote>
<table><caption>TABLE 2. Oligonucleotides used for mutagenesis and PCR assay<sup>a</sup></caption><thead><tr><th>Name</th><th>Target</th><th>Sequence (5' to 3')</th><th>PCR product size (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>iroN.P0</td><td>iroN</td><td>AACTGTGCTCCTGGTTGGGTTGAATAGCCAGGTATCAGTATGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>iroN.P2</td><td>iroN</td><td>AAGCCCGGCCTGGCTCGTTATAGGTATTCGCCCTTCAGACATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>iut P0</td><td>iutA</td><td>GCGCCAGCAGTCGCTCAACAAACCGATGATGAAACGTTCGTGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>iut.P2</td><td>iutA</td><td>CCTGCCTTTGTAGTCGTACAGTGACGCTGGGCCGTAACCCCATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>sit P0</td><td>sit cluster</td><td>GGAACACCGCAGCAGGTACGTAAGGTTGTTGATATAGTTATGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>sit P2</td><td>sit cluster</td><td>TGGATTATATCGCCGATGGTTATCCCGAGCATGTCGCCGACATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>inter P0</td><td></td><td>AGAGCGCGTAGCATTCTTATTTCATGAGGAAATTTACCCGTGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>inter P2</td><td></td><td>CTTGCCTCCTCGCCAGTAATGATTAATGGGAATGCTTCCTCATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>C1</td><td>cat</td><td>TTATACGCAAGGCGACAAGG</td><td></td><td>This study</td></tr><tr><td>C2</td><td>cat</td><td>GATCTTCCGTCACAGGTAGG</td><td></td><td>This study</td></tr><tr><td>iroN.FR1</td><td>iroN</td><td>TATCTGGCAAGGATGTGAGCTTAACGATCA</td><td></td><td>This study</td></tr><tr><td>iroN.FR2</td><td>iroN</td><td>CGCAAATTACACAGCAGGTTGTCATGAGTT</td><td></td><td>This study</td></tr><tr><td>iut.FR1</td><td>iutA</td><td>TTAACTCGCTACACAGCATCTTTGGGCTGA</td><td></td><td>This study</td></tr><tr><td>iut.FR2</td><td>iutA</td><td>TCAGAACAGCACAGAGTAGTTCAGACCAAA</td><td></td><td>This study</td></tr><tr><td>sit FR1p</td><td>sit cluster</td><td>GTGCCTAGTCCTCACCTGCTCGATAGCATT</td><td></td><td>This study</td></tr><tr><td>sit FR1c</td><td>sit cluster</td><td>TAACGCGTCGCCAGCCGGAAACAACCTAAT</td><td></td><td>This study</td></tr><tr><td>sit FR2</td><td>sit cluster</td><td>GCTAATAACAAGTGAACCAGTCCGGCAAT</td><td></td><td>This study</td></tr><tr><td>inter FR1</td><td>Intergenic</td><td>AGGCCATCAGCAAAACACTGATAGTCTGAA</td><td></td><td>This study</td></tr><tr><td>inter FR2</td><td>Intergenic</td><td>AGCTGGTAAATCAGATTTTGCATGGACTGA</td><td></td><td>This study</td></tr><tr><td>p-omp.1</td><td>ompT<sub>P</sub></td><td>GGAAATACAGTAATGCGCCAAT</td><td>189</td><td>This study</td></tr><tr><td>p-omp.2</td><td>ompT<sub>P</sub></td><td>TTAGACGGGTATTCGGATGTTT</td><td>189</td><td>This study</td></tr><tr><td>traJ.1</td><td>traJ<sub>F-plasmid</sub></td><td>GAACTCCCTGCATCGACTGT</td><td>230</td><td>This study</td></tr><tr><td>traJ.2</td><td>traJ<sub>F-plasmid</sub></td><td>CTCTGCGACAGAAGCCATTT</td><td>230</td><td>This study</td></tr><tr><td>ORF143.1</td><td>ORF143</td><td>CTATCGGCTTCCCCTCTTCT</td><td>285</td><td>This study</td></tr><tr><td>ORF143.2</td><td>ORF143</td><td>CTGCATCTCCAGGACTTTGA</td><td>285</td><td>This study</td></tr><tr><td>etsC.1</td><td>etsC</td><td>ATCCCATCAACTGGACCAAG</td><td>359</td><td>This study</td></tr><tr><td>etsC.2</td><td>etsC</td><td>TTCTTCACTGGCATGGACTG</td><td>359</td><td>This study</td></tr><tr><td>cia.1</td><td>cia</td><td>CGCTGGGGTATGATTCAGAT</td><td>456</td><td>This study</td></tr><tr><td>cia.2</td><td>cia</td><td>AGAGCATCCGGCTCTCTGTA</td><td>456</td><td>This study</td></tr><tr><td>cva.F</td><td>cvaA</td><td>ATCCGGGCGTTGTCTGACGGGAAAGTTG</td><td>319</td><td>35</td></tr><tr><td>cva.R</td><td>cvaA</td><td>ACCAGGGAACAGAGGCACCCGGCGTATT</td><td>319</td><td></td></tr><tr><td>issF</td><td>iss</td><td>CAGCAACCCGAACCACTTGATG</td><td>323</td><td>35</td></tr><tr><td>issR</td><td>iss</td><td>AGCATTGCCAGAGCGGCAGAA</td><td>323</td><td></td></tr><tr><td>hlyF.R</td><td>hlyF</td><td>GGCGATTTAGGCATTCCGATACTC</td><td>599</td><td>35</td></tr><tr><td>hlyF.R</td><td>hlyF</td><td>ACGGGGTCGCTAGTTAAGGAG</td><td>599</td><td></td></tr><tr><td>tsh.F</td><td>tsh</td><td>GGGAAATGACCTGAATGCTGG</td><td>420</td><td>35</td></tr><tr><td>tsh.R</td><td>tsh</td><td>CCGCTCATCAGTCAGTACCAC</td><td>420</td><td>35</td></tr><tr><td>eitB-F</td><td>eit</td><td>CAGCAGCGCTTCGGACAAAATCTCCT</td><td>380</td><td>35</td></tr><tr><td>eitB-R</td><td>eit</td><td>TTCCCCACCACTCTCCGTTCTCAAAC</td><td>380</td><td>35</td></tr><tr><td>sitA.F</td><td>sitA</td><td>CGCAGGGGGCACAACTGAT</td><td>663</td><td>52</td></tr><tr><td>sitA.R</td><td>sitA</td><td>CCCTGTACCAGCGTACTGG</td><td>663</td><td></td></tr><tr><td>iroN.1</td><td>iroN</td><td>GAAAGCTCTGGTGGACGGTA</td><td>126</td><td>13</td></tr><tr><td>iroN.2</td><td>iroN</td><td>CGACAGAGGATTACCGGTGT</td><td>126</td><td>13</td></tr><tr><td>aer.1</td><td>iucC</td><td>AAACCTGGTTTACGCAACTGT</td><td>269</td><td>13</td></tr><tr><td>aer.2</td><td>iucC</td><td>ACCCGTCTGCAAATCATGGAT</td><td>269</td><td>13</td></tr></tbody></table>

bromide and photographed with UV transillumination. In each PCR run, a lysate of strain S88 was used as a positive control. Finally, all strains harboring the 11 pS88 genes were also screened for *eitB* and *tsh* genes using the primers described by Johnson et al. (35).

Plasmid sizes were determined by PFGE of undigested DNAs of strains harboring the 11 pS88 genes. PFGE followed by Southern blot hybridization with an *iroN* probe were performed as previously described (13).

**Cure of the pS88 plasmid.** Strain S88 was grown for 18 h in Luria-Bertani (LB) medium at 37°C with shaking. The culture was then diluted to 10<sup>5</sup> CFU/ml, and serial concentrations (2.5, 5, and 10%) of sodium dodecyl sulfate were added. After 18 h of growth with shaking, the cultures were plated on LB agar. After overnight incubation, 200 colonies were screened for colicin production by prick-ing them out on LB agar plates overlaid with a suspension of *E. coli* strain K-12, which is sensitive to colicin. Test strains were assumed to have lost the ColV

<footnote><sup>a</sup> Oligonucleotide primers used for gene recombination are designated by the suffixes P0 and P2; bold characters in the primer sequences indicate the 20 nucleotides homologous to the *cat* gene sequence. The primers used to control the correct introduction of the *cat* gene are designated by the suffixes FR1 and FR2 and flank the DNA target segment. Oligonucleotides designated by sit FR1p and sit FR1c were used to check for the correct introduction of the *cat* gene in the plasmid and chromosomal *sit* loci, respectively. Oligonucleotides designated by inter P0 and inter P2 amplify a noncoding plasmid region located between ORFs 131 and 132. Oligonucleotide primers from p-omp.1 to the end of the table were used to study the molecular epidemiology of the plasmid. *TraJ*<sub>F-plasmid</sub> corresponds to the *traJ* gene of F-type plasmids.</footnote>
<table><caption>TABLE 2. Oligonucleotides used for mutagenesis and PCR assay<sup>a</sup></caption><thead><tr><th>Name</th><th>Target</th><th>Sequence (5' to 3')</th><th>PCR product size (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>iroN.P0</td><td>iroN</td><td>AACTGTGCTCCTGGTTGGGTTGAATAGCCAGGTATCAGTATGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>iroN.P2</td><td>iroN</td><td>AAGCCCGGCCTGGCTCGTTATAGGTATTCGCCCTTCAGACATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>iut P0</td><td>iutA</td><td>GCGCCAGCAGTCGCTCAACAAACCGATGATGAAACGTTCGTGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>iut.P2</td><td>iutA</td><td>CCTGCCTTTGTAGTCGTACAGTGACGCTGGGCCGTAACCCCATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>sit P0</td><td>sit cluster</td><td>GGAACACCGCAGCAGGTACGTAAGGTTGTTGATATAGTTATGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>sit P2</td><td>sit cluster</td><td>TGGATTATATCGCCGATGGTTATCCCGAGCATGTCGCCGACATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>inter P0</td><td></td><td>AGAGCGCGTAGCATTCTTATTTCATGAGGAAATTTACCCGTGTGTA<br>GGCTGGAGCTGCTT</td><td></td><td>This study</td></tr><tr><td>inter P2</td><td></td><td>CTTGCCTCCTCGCCAGTAATGATTAATGGGAATGCTTCCTCATATGA<br>ATATCCTCCTTAG</td><td></td><td>This study</td></tr><tr><td>C1</td><td>cat</td><td>TTATACGCAAGGCGACAAGG</td><td></td><td>This study</td></tr><tr><td>C2</td><td>cat</td><td>GATCTTCCGTCACAGGTAGG</td><td></td><td>This study</td></tr><tr><td>iroN.FR1</td><td>iroN</td><td>TATCTGGCAAGGATGTGAGCTTAACGATCA</td><td></td><td>This study</td></tr><tr><td>iroN.FR2</td><td>iroN</td><td>CGCAAATTACACAGCAGGTTGTCATGAGTT</td><td></td><td>This study</td></tr><tr><td>iut.FR1</td><td>iutA</td><td>TTAACTCGCTACACAGCATCTTTGGGCTGA</td><td></td><td>This study</td></tr><tr><td>iut.FR2</td><td>iutA</td><td>TCAGAACAGCACAGAGTAGTTCAGACCAAA</td><td></td><td>This study</td></tr><tr><td>sit FR1p</td><td>sit cluster</td><td>GTGCCTAGTCCTCACCTGCTCGATAGCATT</td><td></td><td>This study</td></tr><tr><td>sit FR1c</td><td>sit cluster</td><td>TAACGCGTCGCCAGCCGGAAACAACCTAAT</td><td></td><td>This study</td></tr><tr><td>sit FR2</td><td>sit cluster</td><td>GCTAATAACAAGTGAACCAGTCCGGCAAT</td><td></td><td>This study</td></tr><tr><td>inter FR1</td><td>Intergenic</td><td>AGGCCATCAGCAAAACACTGATAGTCTGAA</td><td></td><td>This study</td></tr><tr><td>inter FR2</td><td>Intergenic</td><td>AGCTGGTAAATCAGATTTTGCATGGACTGA</td><td></td><td>This study</td></tr><tr><td>p-omp.1</td><td>ompT<sub>P</sub></td><td>GGAAATACAGTAATGCGCCAAT</td><td>189</td><td>This study</td></tr><tr><td>p-omp.2</td><td>ompT<sub>P</sub></td><td>TTAGACGGGTATTCGGATGTTT</td><td>189</td><td>This study</td></tr><tr><td>traJ.1</td><td>traJ<sub>F-plasmid</sub></td><td>GAACTCCCTGCATCGACTGT</td><td>230</td><td>This study</td></tr><tr><td>traJ.2</td><td>traJ<sub>F-plasmid</sub></td><td>CTCTGCGACAGAAGCCATTT</td><td>230</td><td>This study</td></tr><tr><td>ORF143.1</td><td>ORF143</td><td>CTATCGGCTTCCCCTCTTCT</td><td>285</td><td>This study</td></tr><tr><td>ORF143.2</td><td>ORF143</td><td>CTGCATCTCCAGGACTTTGA</td><td>285</td><td>This study</td></tr><tr><td>etsC.1</td><td>etsC</td><td>ATCCCATCAACTGGACCAAG</td><td>359</td><td>This study</td></tr><tr><td>etsC.2</td><td>etsC</td><td>TTCTTCACTGGCATGGACTG</td><td>359</td><td>This study</td></tr><tr><td>cia.1</td><td>cia</td><td>CGCTGGGGTATGATTCAGAT</td><td>456</td><td>This study</td></tr><tr><td>cia.2</td><td>cia</td><td>AGAGCATCCGGCTCTCTGTA</td><td>456</td><td>This study</td></tr><tr><td>cva.F</td><td>cvaA</td><td>ATCCGGGCGTTGTCTGACGGGAAAGTTG</td><td>319</td><td>35</td></tr><tr><td>cva.R</td><td>cvaA</td><td>ACCAGGGAACAGAGGCACCCGGCGTATT</td><td>319</td><td></td></tr><tr><td>issF</td><td>iss</td><td>CAGCAACCCGAACCACTTGATG</td><td>323</td><td>35</td></tr><tr><td>issR</td><td>iss</td><td>AGCATTGCCAGAGCGGCAGAA</td><td>323</td><td></td></tr><tr><td>hlyF.R</td><td>hlyF</td><td>GGCGATTTAGGCATTCCGATACTC</td><td>599</td><td>35</td></tr><tr><td>hlyF.R</td><td>hlyF</td><td>ACGGGGTCGCTAGTTAAGGAG</td><td>599</td><td></td></tr><tr><td>tsh.F</td><td>tsh</td><td>GGGAAATGACCTGAATGCTGG</td><td>420</td><td>35</td></tr><tr><td>tsh.R</td><td>tsh</td><td>CCGCTCATCAGTCAGTACCAC</td><td>420</td><td>35</td></tr><tr><td>eitB-F</td><td>eit</td><td>CAGCAGCGCTTCGGACAAAATCTCCT</td><td>380</td><td>35</td></tr><tr><td>eitB-R</td><td>eit</td><td>TTCCCCACCACTCTCCGTTCTCAAAC</td><td>380</td><td>35</td></tr><tr><td>sitA.F</td><td>sitA</td><td>CGCAGGGGGCACAACTGAT</td><td>663</td><td>52</td></tr><tr><td>sitA.R</td><td>sitA</td><td>CCCTGTACCAGCGTACTGG</td><td>663</td><td></td></tr><tr><td>iroN.1</td><td>iroN</td><td>GAAAGCTCTGGTGGACGGTA</td><td>126</td><td>13</td></tr><tr><td>iroN.2</td><td>iroN</td><td>CGACAGAGGATTACCGGTGT</td><td>126</td><td>13</td></tr><tr><td>aer.1</td><td>iucC</td><td>AAACCTGGTTTACGCAACTGT</td><td>269</td><td>13</td></tr><tr><td>aer.2</td><td>iucC</td><td>ACCCGTCTGCAAATCATGGAT</td><td>269</td><td>13</td></tr></tbody></table>

bromide and photographed with UV transillumination. In each PCR run, a lysate of strain S88 was used as a positive control. Finally, all strains harboring the 11 pS88 genes were also screened for *eitB* and *tsh* genes using the primers described by Johnson et al. (35).

Plasmid sizes were determined by PFGE of undigested DNAs of strains harboring the 11 pS88 genes. PFGE followed by Southern blot hybridization with an *iroN* probe were performed as previously described (13).

**Cure of the pS88 plasmid.** Strain S88 was grown for 18 h in Luria-Bertani (LB) medium at 37°C with shaking. The culture was then diluted to 10<sup>5</sup> CFU/ml, and serial concentrations (2.5, 5, and 10%) of sodium dodecyl sulfate were added. After 18 h of growth with shaking, the cultures were plated on LB agar. After overnight incubation, 200 colonies were screened for colicin production by prick-ing them out on LB agar plates overlaid with a suspension of *E. coli* strain K-12, which is sensitive to colicin. Test strains were assumed to have lost the ColV

<footnote><sup>a</sup> Oligonucleotide primers used for gene recombination are designated by the suffixes P0 and P2; bold characters in the primer sequences indicate the 20 nucleotides homologous to the *cat* gene sequence. The primers used to control the correct introduction of the *cat* gene are designated by the suffixes FR1 and FR2 and flank the DNA target segment. Oligonucleotides designated by sit FR1p and sit FR1c were used to check for the correct introduction of the *cat* gene in the plasmid and chromosomal *sit* loci, respectively. Oligonucleotides designated by inter P0 and inter P2 amplify a noncoding plasmid region located between ORFs 131 and 132. Oligonucleotide primers from p-omp.1 to the end of the table were used to study the molecular epidemiology of the plasmid. *TraJ*<sub>F-plasmid</sub> corresponds to the *traJ* gene of F-type plasmids.</footnote>
<figure><img src="image_2.png" /></figure>

FIG. 1. Circular representation of the *Escherichia coli* strain S88 plasmid (pS88). Circles display (from the outside) (i) GC percent deviation (GC window – mean GC) in a 1,000-bp window, (ii) predicted ORFs transcribed in the clockwise direction, (iii) predicted ORFs transcribed in the counterclockwise direction, (iv) GC skew (G + C/G – C) in a 1,000-bp window, (v) transposable elements (pink), and (vi) coordinates in kilobase pairs (kbp) from the origin of replication. Genes displayed in circles ii and iii are categorized by color as follows: red, iron uptake systems; orange, other putative virulence factors; yellow, bacteriocin production and immunity; pink, mobile genetic elements; dark blue, plasmid transfer; green, plasmid replication; teal, plasmid maintenance; gray, unknown.

plasmid if they were unable to inhibit the growth of strain K-12. This was confirmed, using the PCRs described above, by the loss of the 11 plasmid-related ORFs or genes and by the disappearance of extrachromosomal DNA on agarose gel electrophoresis after plasmid preparation with the relevant colonies (Qiaprep Minispin, Qiagen). For each variant thus obtained, we checked the expression of the K1 capsule antigen and the presence of chromosomal virulence genes, using multiplex PCR as previously described (14).

**Mutant construction.** S88 mutants were obtained with the PCR-based method of Datsenko and Wanner (19) as previously described, with plasmids kindly provided by Lionello and Nara Bossi (Centre de Génétique Moléculaire, CNRS, Gif sur Yvette, France) (47, 50). The primers used for insertional mutation are listed in Table 2. Correct introduction of the *cat* gene into the target was controlled by PCR using primers homologous to the *cat* gene and the flanking region of the target, as previously described (47, 50). For each mutant thus obtained, we checked the expression of the K1 capsule antigen, bacteriocin production, the presence of nondeleted plasmid genes, and chromosomal virulence genes, using multiplex PCRs.

**Conjugal plasmid transfer.** The *cat* gene was introduced into a noncoding region of pS88 between ORFs 131 and 132, using the method of Datsenko and Wanner (19), so that the plasmid harbored a chloramphenicol resistance determinant (plasmid pS88cat) for the selection of transconjugant clones after conjugal transfer. The cured S88 variant was made resistant to nalidixic acid in order to select transconjugants for pS88cat reintroduction.

Donor and recipient cells were grown overnight at 37°C in LB broth. A 10⁻² dilution was prepared with fresh LB medium. After incubation at 37°C for 2.5 h without shaking, 2.5-ml aliquots of each culture were mixed. After overnight incubation, the mating mixture was harvested and bacteria were plated on selective medium. The transconjugants thus obtained were checked for colicin production and the presence of plasmid-related genes by PCR and by gel electrophoresis of a plasmid extract (Qiaprep minispin; Qiagen).

**Experimental models.** We assessed the ability of the wild-type strain and mutants to induce high-level bacteremia in newborn rats, as previously described (30). Briefly, pathogen-free Sprague-Dawley rats were obtained from Charles River Laboratories at 4 days of age, with their mothers. At 5 days of age, the pups

were inoculated intraperitoneally with a normal saline suspension containing ~500 CFU of the test strain. A tail incision was made 18 h after inoculation, and 5 µl of blood was sampled. Serial dilutions were plated for CFU counting. Comparisons of bacterial counts in the animal model were based on a two-sample unpaired *t* test. Data are expressed as mean ± standard deviation. *P* values below 0.05 were considered to denote statistically significant differences.

Serum bactericidal activity against *E. coli* strains was determined using pooled sera of healthy volunteers as previously described (30). To measure bactericidal serum activity, 20 µl of a bacterial inoculum of 10⁸ CFU/ml in physiological serum was added to 180 µl of freshly thawed pure serum. Quantitative cultures were done 5 h later. Experiments were repeated five times.

**Nucleotide sequence accession number.** The DNA sequence of pS88 has been deposited in GenBank under accession number CU928146.

## RESULTS

**Sequencing of pS88.** (i) Overview. A complete circularized DNA sequence of plasmid pS88 was obtained. It was 133,853 bp long, with an overall G+C content of 49.28% (Fig. 1). A total of 133 protein-coding genes plus 11 fragments of protein-coding genes corresponding to six pseudogenes were identified in the sequence (Table 3). Globally, based on sequence homologies, the plasmid could be separated into two halves. The first, from base 20,000 to base 86,000, is mostly composed of the genes involved in plasmid-related functions (conjugal transfer, maintenance, and partition); all are almost exclusively transcribed in the clockwise direction, with an average G+C content of 51%. The other half contains most of the putative or known virulence-associated genes, such as those encoding iron uptake systems and factors involved in resistance to the innate
<figure><img src="image_3.png" /></figure>

FIG. 1. Circular representation of the *Escherichia coli* strain S88 plasmid (pS88). Circles display (from the outside) (i) GC percent deviation (GC window – mean GC) in a 1,000-bp window, (ii) predicted ORFs transcribed in the clockwise direction, (iii) predicted ORFs transcribed in the counterclockwise direction, (iv) GC skew (G + C/G – C) in a 1,000-bp window, (v) transposable elements (pink), and (vi) coordinates in kilobase pairs (kbp) from the origin of replication. Genes displayed in circles ii and iii are categorized by color as follows: red, iron uptake systems; orange, other putative virulence factors; yellow, bacteriocin production and immunity; pink, mobile genetic elements; dark blue, plasmid transfer; green, plasmid replication; teal, plasmid maintenance; gray, unknown.

plasmid if they were unable to inhibit the growth of strain K-12. This was confirmed, using the PCRs described above, by the loss of the 11 plasmid-related ORFs or genes and by the disappearance of extrachromosomal DNA on agarose gel electrophoresis after plasmid preparation with the relevant colonies (Qiaprep Minispin, Qiagen). For each variant thus obtained, we checked the expression of the K1 capsule antigen and the presence of chromosomal virulence genes, using multiplex PCR as previously described (14).

**Mutant construction.** S88 mutants were obtained with the PCR-based method of Datsenko and Wanner (19) as previously described, with plasmids kindly provided by Lionello and Nara Bossi (Centre de Génétique Moléculaire, CNRS, Gif sur Yvette, France) (47, 50). The primers used for insertional mutation are listed in Table 2. Correct introduction of the *cat* gene into the target was controlled by PCR using primers homologous to the *cat* gene and the flanking region of the target, as previously described (47, 50). For each mutant thus obtained, we checked the expression of the K1 capsule antigen, bacteriocin production, the presence of nondeleted plasmid genes, and chromosomal virulence genes, using multiplex PCRs.

**Conjugal plasmid transfer.** The *cat* gene was introduced into a noncoding region of pS88 between ORFs 131 and 132, using the method of Datsenko and Wanner (19), so that the plasmid harbored a chloramphenicol resistance determinant (plasmid pS88cat) for the selection of transconjugant clones after conjugal transfer. The cured S88 variant was made resistant to nalidixic acid in order to select transconjugants for pS88cat reintroduction.

Donor and recipient cells were grown overnight at 37°C in LB broth. A 10⁻² dilution was prepared with fresh LB medium. After incubation at 37°C for 2.5 h without shaking, 2.5-ml aliquots of each culture were mixed. After overnight incubation, the mating mixture was harvested and bacteria were plated on selective medium. The transconjugants thus obtained were checked for colicin production and the presence of plasmid-related genes by PCR and by gel electrophoresis of a plasmid extract (Qiaprep minispin; Qiagen).

**Experimental models.** We assessed the ability of the wild-type strain and mutants to induce high-level bacteremia in newborn rats, as previously described (30). Briefly, pathogen-free Sprague-Dawley rats were obtained from Charles River Laboratories at 4 days of age, with their mothers. At 5 days of age, the pups

were inoculated intraperitoneally with a normal saline suspension containing ~500 CFU of the test strain. A tail incision was made 18 h after inoculation, and 5 µl of blood was sampled. Serial dilutions were plated for CFU counting. Comparisons of bacterial counts in the animal model were based on a two-sample unpaired *t* test. Data are expressed as mean ± standard deviation. *P* values below 0.05 were considered to denote statistically significant differences.

Serum bactericidal activity against *E. coli* strains was determined using pooled sera of healthy volunteers as previously described (30). To measure bactericidal serum activity, 20 µl of a bacterial inoculum of 10⁸ CFU/ml in physiological serum was added to 180 µl of freshly thawed pure serum. Quantitative cultures were done 5 h later. Experiments were repeated five times.

**Nucleotide sequence accession number.** The DNA sequence of pS88 has been deposited in GenBank under accession number CU928146.

## RESULTS

**Sequencing of pS88.** (i) Overview. A complete circularized DNA sequence of plasmid pS88 was obtained. It was 133,853 bp long, with an overall G+C content of 49.28% (Fig. 1). A total of 133 protein-coding genes plus 11 fragments of protein-coding genes corresponding to six pseudogenes were identified in the sequence (Table 3). Globally, based on sequence homologies, the plasmid could be separated into two halves. The first, from base 20,000 to base 86,000, is mostly composed of the genes involved in plasmid-related functions (conjugal transfer, maintenance, and partition); all are almost exclusively transcribed in the clockwise direction, with an average G+C content of 51%. The other half contains most of the putative or known virulence-associated genes, such as those encoding iron uptake systems and factors involved in resistance to the innate
<table><caption>TABLE 3. Predicted ORFs identified on the DNA sequence of plasmid pS88 with their homologs on plasmids pAPEC-O2-ColV and pAPEC-O1-ColIBM</caption><thead><tr><th rowspan="2">Beginning</th><th rowspan="2">End</th><th rowspan="2">Gene</th><th rowspan="2">ORF</th><th rowspan="2">Product</th><th colspan="2">pAPEC-O2-ColV homolog</th><th colspan="2">pAPEC-O1-ColIBM homolog</th></tr><tr><th>Name</th><th>Identity (%)</th><th>Name</th><th>Identity (%)</th></tr></thead><tbody><tr><td>132</td><td>872</td><td>int</td><td>0001</td><td>Putative site-specific recombinase</td><td>O2ColV23</td><td>100</td><td>O1CoBM187</td><td>99.59</td></tr><tr><td>1157</td><td>2134</td><td>repA</td><td>0002</td><td>RepFIB replication protein RepA</td><td>O2ColV22</td><td>100</td><td>O1CoBM186</td><td>100</td></tr><tr><td>2543</td><td>2839</td><td></td><td>0003</td><td>Conserved hypothetical protein</td><td>O2ColV21</td><td>100</td><td>O1CoBM185</td><td>100</td></tr><tr><td>2808</td><td>3029</td><td></td><td>0004</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>3244</td><td>3309</td><td></td><td>0005</td><td>Putative transposase (fragment) IS21 family</td><td></td><td></td><td></td><td></td></tr><tr><td>3494</td><td>3883</td><td></td><td>0006</td><td>Putative fragment of ImpB UV protection protein</td><td>O2ColV19</td><td>100</td><td>O1CoBM183</td><td>100</td></tr><tr><td>4063</td><td>4566</td><td></td><td>0007</td><td>Transposase InsB (ORF2) ISJ</td><td>O2ColV18</td><td>100</td><td>O1CoBM2</td><td>100</td></tr><tr><td>4485</td><td>4709</td><td>iiuA</td><td>0008</td><td>Transposase InsA (ORF1) ISJ</td><td>O2ColV17</td><td>100</td><td>O1CoBM159</td><td>100</td></tr><tr><td>5285</td><td>7486</td><td>iiuA</td><td>0009</td><td>Ferric aerobactin receptor precursor lusA</td><td>O2ColV15</td><td>99.9</td><td>O1CoBM168</td><td>100</td></tr><tr><td>7568</td><td>8845</td><td>iiuD</td><td>0010</td><td>l-Lysine 6-monooxygenase lusD</td><td>O2ColV14</td><td>99.8</td><td>O1CoBM169</td><td>99.29</td></tr><tr><td>8842</td><td>10584</td><td>iiuC</td><td>0011</td><td>Aerobactin siderophore biosynthesis protein IucC</td><td>O2ColV13</td><td>100</td><td>O1CoBM170</td><td>100</td></tr><tr><td>10584</td><td>11531</td><td>iiuB</td><td>0012</td><td>N(6)-Hydroxylsine acetylase IuxB</td><td>O2ColV12</td><td>99.7</td><td>O1CoBM171</td><td>100</td></tr><tr><td>11532</td><td>13319</td><td>iiuA</td><td>0013</td><td>Aerobactin siderophore biosynthesis protein IucA</td><td>O2ColV11</td><td>99.7</td><td>O1CoBM172</td><td>100</td></tr><tr><td>13392</td><td>14585</td><td>siif</td><td>0014</td><td>Putative membrane transport protein Siif</td><td>O2ColV9</td><td>92</td><td>O1CoBM173</td><td>100</td></tr><tr><td>14965</td><td>15345</td><td></td><td>0015</td><td>Putative membrane protein; CrcB-like protein</td><td>O2ColV8</td><td>97.7</td><td>O1CoBM174</td><td>100</td></tr><tr><td>15417</td><td>15689</td><td></td><td>0016</td><td>Conserved hypothetical protein</td><td>O2ColV7</td><td>100</td><td>O1CoBM175</td><td>100</td></tr><tr><td>15686</td><td>16120</td><td>siifD</td><td>0017</td><td>Putative enolase</td><td>O2ColV6</td><td>100</td><td>O1CoBM176</td><td>100</td></tr><tr><td>16588</td><td>17445</td><td>siifC</td><td>0019</td><td>SiifD protein, iron transport protein, inner membrane component</td><td>O2ColV4</td><td>100</td><td>O1CoBM177</td><td>100</td></tr><tr><td>17442</td><td>18299</td><td>siifC</td><td>0020</td><td>SiifC protein, iron transport protein, inner membrane component</td><td>O2ColV3</td><td>99.2</td><td>O1CoBM178</td><td>99.3</td></tr><tr><td>18296</td><td>19123</td><td>siifB</td><td>0021</td><td>SiifB protein, iron transport protein, ATP-binding component</td><td>O2ColV2</td><td>100</td><td>O1CoBM179</td><td>99.64</td></tr><tr><td>19123</td><td>20057</td><td>siifA</td><td>0022</td><td>SiifA protein, iron transport protein, periplasmic-binding protein</td><td>O2ColV1</td><td>99</td><td>O1CoBM180</td><td>100</td></tr><tr><td>20160</td><td>20051</td><td></td><td>0023</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>20393</td><td>20668</td><td>insA</td><td>0024</td><td>ISJ repressor protein InsA</td><td>O2ColV182</td><td>100</td><td>O1CoBM164</td><td>100</td></tr><tr><td>20695</td><td>21090</td><td>insB</td><td>0025</td><td>Fragment of transposase (partial)</td><td>O2ColV181</td><td>100</td><td></td><td></td></tr><tr><td>21101</td><td>21775</td><td></td><td>0026</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>21933</td><td>22127</td><td></td><td>0027</td><td>Hypothetical protein; putative exported protein</td><td></td><td></td><td></td><td></td></tr><tr><td>22203</td><td>22682</td><td></td><td>0028</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>22800</td><td>23249</td><td></td><td>0029</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>23866</td><td>25368</td><td></td><td>0030</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>25594</td><td>25785</td><td></td><td>0031</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>27444</td><td>27668</td><td></td><td>0035</td><td>Transposase InsA (ORF1) ISJ</td><td>O2ColV197</td><td>98.4</td><td>O1CoBM163</td><td>99.37</td></tr><tr><td>27587</td><td>28090</td><td>insB</td><td>0036</td><td>Transposase</td><td>O2ColV198</td><td>97.8</td><td>O1CoBM162</td><td>100</td></tr><tr><td>28381</td><td>29556</td><td>sopA</td><td>0037</td><td>SopA protein (plasmid partition protein A)</td><td>O2ColV199</td><td>99</td><td>O1CoBM160</td><td>99.8</td></tr><tr><td>29556</td><td>30527</td><td>sopB</td><td>0038</td><td>SopB protein (plasmid partition protein B)</td><td>O2ColV161</td><td>26.5</td><td></td><td></td></tr><tr><td>31055</td><td>31216</td><td></td><td>0039</td><td>Hypothetical protein</td><td>O2ColV160</td><td>25.3</td><td>O1CoBM3</td><td>100</td></tr><tr><td>31244</td><td>31591</td><td></td><td>0040</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>31654</td><td>32235</td><td></td><td>0041</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>32721</td><td>33692</td><td></td><td>0042</td><td>Transposase ISJ10 family</td><td>O2ColV185</td><td>25</td><td>O1CoBM4</td><td>100</td></tr><tr><td>33844</td><td>34014</td><td>yubI</td><td>0043</td><td>Hypothetical protein</td><td>O2ColV83</td><td>28.9</td><td></td><td></td></tr><tr><td>34073</td><td>34519</td><td></td><td>0044</td><td>Putative antirestriction protein</td><td></td><td></td><td></td><td></td></tr><tr><td>34609</td><td>34980</td><td></td><td>0045</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>34980</td><td>35162</td><td></td><td>0046</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>36438</td><td>37799</td><td>yubA</td><td>0047</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>37847</td><td>38410</td><td>ydcA</td><td>0048</td><td>Putative adenine-specific DNA methylase</td><td></td><td></td><td></td><td></td></tr><tr><td>39216</td><td>39848</td><td>ssb</td><td>0050</td><td>Single-stranded DNA-binding protein</td><td></td><td></td><td></td><td></td></tr><tr><td>39784</td><td>40044</td><td>yubL</td><td>0051</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>40381</td><td>41922</td><td></td><td>0052</td><td>Putative transposase ORF1, IS21 family</td><td>O2ColV72</td><td>46.8</td><td>O1CoBM177</td><td>100</td></tr><tr><td>41934</td><td>42683</td><td></td><td>0053</td><td>Putative transposase ORF2, IS21 family</td><td>O2ColV73</td><td>45.3</td><td>O1CoBM76</td><td>100</td></tr><tr><td>42799</td><td>44652</td><td>ycjA</td><td>0054</td><td>Putative DNA-binding protein involved in plasmid partitioning (ParB-like partition protein)</td><td></td><td></td><td></td><td></td></tr><tr><td>44704</td><td>45141</td><td>psiB</td><td>0055</td><td>Plasmid SOS inhibition protein B</td><td>O2ColV145</td><td>97.2</td><td>O1CoBM17</td><td>99.31</td></tr><tr><td>45138</td><td>45857</td><td>psiA</td><td>0056</td><td>Plasmid SOS inhibition protein A</td><td>O2ColV144</td><td>97.5</td><td>O1CoBM18</td><td>97.49</td></tr><tr><td>45991</td><td>46290</td><td>fmnC</td><td>0057</td><td>Putative F-plasmid maintenance protein C</td><td></td><td></td><td></td><td></td></tr><tr><td>46532</td><td>46849</td><td>yubN</td><td>0059</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>47193</td><td>47480</td><td>yubO</td><td>0060</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>47518</td><td>48420</td><td>yubP</td><td>0061</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>48716</td><td>49225</td><td>yubQ</td><td>0062</td><td>X polypeptide (P19 protein); putative transglycosylase</td><td></td><td></td><td></td><td></td></tr><tr><td>49640</td><td>50023</td><td>traM</td><td>0063</td><td>Protein TraM (conjugal transfer protein M)</td><td></td><td></td><td></td><td></td></tr><tr><td>50129</td><td>50899</td><td>traD</td><td>0064</td><td>Protein TraJ (positive regulator of conjugal transfer operon)</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>TABLE 3. Predicted ORFs identified on the DNA sequence of plasmid pS88 with their homologs on plasmids pAPEC-O2-ColV and pAPEC-O1-ColIBM</caption><thead><tr><th rowspan="2">Beginning</th><th rowspan="2">End</th><th rowspan="2">Gene</th><th rowspan="2">ORF</th><th rowspan="2">Product</th><th colspan="2">pAPEC-O2-ColV homolog</th><th colspan="2">pAPEC-O1-ColIBM homolog</th></tr><tr><th>Name</th><th>Identity (%)</th><th>Name</th><th>Identity (%)</th></tr></thead><tbody><tr><td>132</td><td>872</td><td>int</td><td>0001</td><td>Putative site-specific recombinase</td><td>O2ColV23</td><td>100</td><td>O1CoBM187</td><td>99.59</td></tr><tr><td>1157</td><td>2134</td><td>repA</td><td>0002</td><td>RepFIB replication protein RepA</td><td>O2ColV22</td><td>100</td><td>O1CoBM186</td><td>100</td></tr><tr><td>2543</td><td>2839</td><td></td><td>0003</td><td>Conserved hypothetical protein</td><td>O2ColV21</td><td>100</td><td>O1CoBM185</td><td>100</td></tr><tr><td>2808</td><td>3029</td><td></td><td>0004</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>3244</td><td>3309</td><td></td><td>0005</td><td>Putative transposase (fragment) IS21 family</td><td></td><td></td><td></td><td></td></tr><tr><td>3494</td><td>3883</td><td></td><td>0006</td><td>Putative fragment of ImpB UV protection protein</td><td>O2ColV19</td><td>100</td><td>O1CoBM183</td><td>100</td></tr><tr><td>4063</td><td>4566</td><td></td><td>0007</td><td>Transposase InsB (ORF2) ISJ</td><td>O2ColV18</td><td>100</td><td>O1CoBM2</td><td>100</td></tr><tr><td>4485</td><td>4709</td><td>iiuA</td><td>0008</td><td>Transposase InsA (ORF1) ISJ</td><td>O2ColV17</td><td>100</td><td>O1CoBM159</td><td>100</td></tr><tr><td>5285</td><td>7486</td><td>iiuA</td><td>0009</td><td>Ferric aerobactin receptor precursor lusA</td><td>O2ColV15</td><td>99.9</td><td>O1CoBM168</td><td>100</td></tr><tr><td>7568</td><td>8845</td><td>iiuD</td><td>0010</td><td>l-Lysine 6-monooxygenase IucD</td><td>O2ColV14</td><td>99.8</td><td>O1CoBM169</td><td>99.29</td></tr><tr><td>8842</td><td>10584</td><td>iiuC</td><td>0011</td><td>Aerobactin siderophore biosynthesis protein IucC</td><td>O2ColV13</td><td>100</td><td>O1CoBM170</td><td>100</td></tr><tr><td>10584</td><td>11531</td><td>iiuB</td><td>0012</td><td>N(6)-Hydroxylsine acetylase IucB</td><td>O2ColV12</td><td>99.7</td><td>O1CoBM171</td><td>100</td></tr><tr><td>11532</td><td>13319</td><td>iiuA</td><td>0013</td><td>Aerobactin siderophore biosynthesis protein IucA</td><td>O2ColV11</td><td>99.7</td><td>O1CoBM172</td><td>100</td></tr><tr><td>13392</td><td>14585</td><td>siif</td><td>0014</td><td>Putative membrane transport protein Siif</td><td>O2ColV9</td><td>92</td><td>O1CoBM173</td><td>100</td></tr><tr><td>14965</td><td>15345</td><td></td><td>0015</td><td>Putative membrane protein; CrcB-like protein</td><td>O2ColV8</td><td>97.7</td><td>O1CoBM174</td><td>100</td></tr><tr><td>15417</td><td>15689</td><td></td><td>0016</td><td>Conserved hypothetical protein</td><td>O2ColV7</td><td>100</td><td>O1CoBM175</td><td>100</td></tr><tr><td>15686</td><td>16120</td><td>siitD</td><td>0017</td><td>Putative enolase</td><td>O2ColV6</td><td>100</td><td>O1CoBM176</td><td>100</td></tr><tr><td>16588</td><td>17445</td><td>siitC</td><td>0019</td><td>SiitD protein, iron transport protein, inner membrane component</td><td>O2ColV4</td><td>100</td><td>O1CoBM177</td><td>100</td></tr><tr><td>17442</td><td>18299</td><td>siitC</td><td>0020</td><td>SiitC protein, iron transport protein, inner membrane component</td><td>O2ColV3</td><td>99.2</td><td>O1CoBM178</td><td>99.3</td></tr><tr><td>18296</td><td>19123</td><td>siitB</td><td>0021</td><td>SiitB protein, iron transport protein, ATP-binding component</td><td>O2ColV2</td><td>100</td><td>O1CoBM179</td><td>99.64</td></tr><tr><td>19123</td><td>20057</td><td>siitA</td><td>0022</td><td>SiitA protein, iron transport protein, periplasmic-binding protein</td><td>O2ColV1</td><td>99</td><td>O1CoBM180</td><td>100</td></tr><tr><td>20160</td><td>20051</td><td></td><td>0023</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>20393</td><td>20668</td><td>insA</td><td>0024</td><td>ISJ repressor protein InsA</td><td>O2ColV182</td><td>100</td><td>O1CoBM164</td><td>100</td></tr><tr><td>20695</td><td>21090</td><td>insB</td><td>0025</td><td>Fragment of transposase (partial)</td><td>O2ColV181</td><td>100</td><td></td><td></td></tr><tr><td>21101</td><td>21775</td><td></td><td>0026</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>21933</td><td>22127</td><td></td><td>0027</td><td>Hypothetical protein; putative exported protein</td><td></td><td></td><td></td><td></td></tr><tr><td>22203</td><td>22682</td><td></td><td>0028</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>22800</td><td>23249</td><td></td><td>0029</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>23866</td><td>25368</td><td></td><td>0030</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>25594</td><td>25785</td><td></td><td>0031</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>27444</td><td>27668</td><td></td><td>0035</td><td>Transposase InsA (ORF1) ISJ</td><td>O2ColV197</td><td>98.4</td><td>O1CoBM163</td><td>99.37</td></tr><tr><td>27587</td><td>28090</td><td>insB</td><td>0036</td><td>Transposase</td><td>O2ColV198</td><td>97.8</td><td>O1CoBM162</td><td>100</td></tr><tr><td>28381</td><td>29556</td><td>sopA</td><td>0037</td><td>SopA protein (plasmid partition protein A)</td><td>O2ColV199</td><td>99</td><td>O1CoBM160</td><td>99.8</td></tr><tr><td>29556</td><td>30527</td><td>sopB</td><td>0038</td><td>SopB protein (plasmid partition protein B)</td><td>O2ColV161</td><td>26.5</td><td></td><td></td></tr><tr><td>31055</td><td>31216</td><td></td><td>0039</td><td>Hypothetical protein</td><td>O2ColV160</td><td>25.3</td><td>O1CoBM3</td><td>100</td></tr><tr><td>31244</td><td>31591</td><td></td><td>0040</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>31654</td><td>32235</td><td></td><td>0041</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>32721</td><td>33692</td><td></td><td>0042</td><td>Transposase ISJ10 family</td><td>O2ColV185</td><td>25</td><td>O1CoBM4</td><td>100</td></tr><tr><td>33844</td><td>34014</td><td>yubI</td><td>0043</td><td>Hypothetical protein</td><td>O2ColV83</td><td>28.9</td><td></td><td></td></tr><tr><td>34073</td><td>34519</td><td></td><td>0044</td><td>Putative antirestriction protein</td><td></td><td></td><td></td><td></td></tr><tr><td>34609</td><td>34980</td><td></td><td>0045</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>34980</td><td>35162</td><td></td><td>0046</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>36438</td><td>37799</td><td>yubA</td><td>0047</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>37847</td><td>38410</td><td>ydcA</td><td>0048</td><td>Putative adenine-specific DNA methylase</td><td></td><td></td><td></td><td></td></tr><tr><td>39216</td><td>39848</td><td>ssb</td><td>0050</td><td>Single-stranded DNA-binding protein</td><td></td><td></td><td></td><td></td></tr><tr><td>39784</td><td>40044</td><td>yubL</td><td>0051</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>40381</td><td>41922</td><td></td><td>0052</td><td>Putative transposase ORF1, IS21 family</td><td>O2ColV72</td><td>46.8</td><td>O1CoBM177</td><td>100</td></tr><tr><td>41934</td><td>42683</td><td></td><td>0053</td><td>Putative transposase ORF2, IS21 family</td><td>O2ColV73</td><td>45.3</td><td>O1CoBM76</td><td>100</td></tr><tr><td>42799</td><td>44652</td><td>ycjA</td><td>0054</td><td>Putative DNA-binding protein involved in plasmid partitioning (ParB-like partition protein)</td><td></td><td></td><td></td><td></td></tr><tr><td>44704</td><td>45141</td><td>psiB</td><td>0055</td><td>Plasmid SOS inhibition protein B</td><td>O2ColV145</td><td>97.2</td><td>O1CoBM17</td><td>99.31</td></tr><tr><td>45138</td><td>45857</td><td>psiA</td><td>0056</td><td>Plasmid SOS inhibition protein A</td><td>O2ColV144</td><td>97.5</td><td>O1CoBM18</td><td>97.49</td></tr><tr><td>45991</td><td>46290</td><td>fmnC</td><td>0057</td><td>Putative F-plasmid maintenance protein C</td><td></td><td></td><td></td><td></td></tr><tr><td>46532</td><td>46849</td><td>yubN</td><td>0059</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>47193</td><td>47480</td><td>yubO</td><td>0060</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>47518</td><td>48420</td><td>yubP</td><td>0061</td><td>Conserved hypothetical protein</td><td></td><td></td><td></td><td></td></tr><tr><td>48716</td><td>49225</td><td>yubQ</td><td>0062</td><td>X polypeptide (P19 protein); putative transglycosylase</td><td></td><td></td><td></td><td></td></tr><tr><td>49640</td><td>50023</td><td>traM</td><td>0063</td><td>Protein TraM (conjugal transfer protein M)</td><td></td><td></td><td></td><td></td></tr><tr><td>50129</td><td>50899</td><td>traD</td><td>0064</td><td>Protein TraJ (positive regulator of conjugal transfer operon)</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>TABLE 3—Continued</caption><thead><tr><th colspan="2">Position</th><th rowspan="2">ORF</th><th rowspan="2">Product</th><th colspan="2">pAPEC-O2-ColV homolog</th><th colspan="2">pAPEC-O1-ColBM homolog</th></tr><tr><th>Beginning</th><th>End</th><th>Name</th><th>Identity (%)</th><th>Name</th><th>Identity (%)</th></tr></thead><tbody><tr><td>110582</td><td>111217</td><td></td><td>Conserved hypothetical protein</td><td>O2ColV51</td><td>95.6</td><td>O1CoBM143</td><td>100</td></tr><tr><td>111327</td><td>111632</td><td></td><td>Conserved hypothetical protein</td><td>O2ColV50</td><td>99</td><td>O1CoBM144</td><td>100</td></tr><tr><td>112196</td><td>112378</td><td>iss</td><td>Hypothetical protein</td><td>O2ColV49.2</td><td>100</td><td>O1CoBM146</td><td>100</td></tr><tr><td>112492</td><td>112785</td><td>yqfG</td><td>Iss (Increased serum survival)</td><td>O2ColV49</td><td>100</td><td>O1CoBM126</td><td>100</td></tr><tr><td>113412</td><td>113777</td><td></td><td>Transposase ORF A, IS2</td><td>O2ColV48.2</td><td>99.6</td><td>O1CoBM149</td><td>100</td></tr><tr><td>113735</td><td>114640</td><td>yqfG</td><td>Transposase ORF 2, IS2</td><td>O2ColV48</td><td>97.3</td><td>O1CoBM150</td><td>100</td></tr><tr><td>115306</td><td>115449</td><td></td><td>Hypothetical protein</td><td></td><td></td><td>O1CoBM151</td><td>99.75</td></tr><tr><td>115864</td><td>117057</td><td></td><td>Conserved hypothetical protein; putative GTPase</td><td>O2ColV47</td><td>100</td><td>O1CoBM152</td><td>100</td></tr><tr><td>117190</td><td>117507</td><td></td><td>Putative transposase ORF B (fragment), IS2</td><td>O2ColV46</td><td>100</td><td>O1CoBM153</td><td>100</td></tr><tr><td>117694</td><td>118560</td><td></td><td>Putative transposase (fragment)</td><td>O2ColV45</td><td>95.8</td><td></td><td></td></tr><tr><td>118538</td><td>118648</td><td></td><td>Putative transposase (fragment)</td><td>O2ColV45</td><td>83.9</td><td></td><td></td></tr><tr><td>118697</td><td>119083</td><td></td><td>Putative transposase (fragment)</td><td>O2ColV45</td><td>93</td><td></td><td></td></tr><tr><td>119120</td><td>119308</td><td></td><td>Conserved hypothetical protein</td><td></td><td></td><td>O1CoBM154</td><td>100</td></tr><tr><td>119318</td><td>120259</td><td>etsC</td><td>Conserved hypothetical protein</td><td></td><td></td><td>O1CoBM155</td><td>100</td></tr><tr><td>121056</td><td>122426</td><td>etsB</td><td>Putative type I secretion outer membrane protein EtsC</td><td>O2ColV35</td><td>100</td><td>O1CoBM156</td><td>100</td></tr><tr><td>122430</td><td>124370</td><td></td><td>Putative type I secretion ATP-binding protein EtsB</td><td>O2ColV34</td><td>99.9</td><td>O1CoBM199</td><td>100</td></tr><tr><td>124367</td><td>125575</td><td>etsA</td><td>Putative type I secretion membrane-fusion protein EtsA</td><td>O2ColV33</td><td>98.7</td><td>O1CoBM198</td><td>99.85</td></tr><tr><td>126448</td><td>126738</td><td></td><td>Putative transposase ORF B (fragment), IS3 family</td><td></td><td></td><td>O1CoBM197</td><td>99.75</td></tr><tr><td>126687</td><td>126860</td><td></td><td>Putative transposase ORF B (fragment), IS3 family</td><td></td><td></td><td>O1CoBM196</td><td>100</td></tr><tr><td>127008</td><td>127898</td><td></td><td>Putative transposase ORF B, IS3 family, IS57 group</td><td>O2ColV95</td><td>99.2</td><td>O1CoBM194</td><td>100</td></tr><tr><td>127895</td><td>128221</td><td></td><td>Transposase ORF A, IS3 family, IS37 group</td><td>O2ColV96</td><td>93.5</td><td>O1CoBM192</td><td>100</td></tr><tr><td>128405</td><td>128563</td><td></td><td>Putative transposase ORF B (fragment), IS family, IS407 group</td><td>O2ColV29</td><td>98.1</td><td>O1CoBM154</td><td>100</td></tr><tr><td>129343</td><td>129732</td><td></td><td>Hypothetical protein</td><td>O2ColV27</td><td>96.9</td><td>O1CoBM155</td><td>100</td></tr><tr><td>129836</td><td>130789</td><td>ompT<sub>P</sub></td><td>Outer membrane protease (omptin)</td><td>O2ColV26</td><td>100</td><td></td><td></td></tr><tr><td>131222</td><td>132331</td><td>hlyF</td><td>Hemolysin HlyF</td><td>O2ColV24</td><td>100</td><td>O1CoBM189</td><td>100</td></tr><tr><td>132394</td><td>133302</td><td></td><td>Conserved hypothetical protein; putative Mlg-14 protein</td><td></td><td></td><td>O1CoBM188</td><td>99.67</td></tr></tbody></table>

immune system (see below); they were transcribed in both directions and showed sharp deviations from the average G+C content (29.9% to 63.6%), suggesting a foreign mosaic origin. Thirty-five ORFs of unknown function and 14 insertion sequence-like genes were scattered throughout the plasmid. Two different colicins, colicin Ia and colicin V, along with their immunity protein, were encoded by pS88.

(ii) Replication, transfer, and maintenance regions of pS88. Plasmid pS88 harbors two replicons. The first replicon region, RepFIB, contains the typical replication gene *repA* and the site-specific integrase *int* (55). The second, RepFIIA, encodes the CopB repressor and the RepA1 replication proteins. Thus, pS88 appears to belong to both the IncFI and IncFII incompatibility groups. The complete F-like transfer region of pS88 spans 31,485 bp and contains 32 genes (from *traM* to *finO*). The DNA sequences of the three regulator genes *traM*, *traJ*, and *traY*, relative to publicly available databases, appear to be characteristic of an F-type plasmid rather than an R1-type plasmid (15) (Fig. 2). None of the other *tra* genes were discriminated (as exemplified by *traT*), except for *traS*, which was closer to that of R1-type plasmids (Fig. 2). Upstream of the transfer locus lie genes involved in single-stranded DNA transfer; *ssb*, the gene encoding single-stranded binding protein; and *psiA* and *psiB*, plasmid SOS inhibition genes (6). Two loci putatively involved in plasmid maintenance were identified. One, close to the transfer region, was composed of the *sopA* and *sopB* genes, coding for the plasmid partition proteins (45). Located downstream of the RepFIIA replicon, the *yacABC* operon may represent a toxin-antitoxin plasmid stability system of which a second copy, albeit truncated, lies between the colicin V and salmochelin operons.

(iii) Putative virulence region of S88. The putative virulence region of pS88 harbored three different iron uptake systems, namely, aerobactin (*iucABCD* and *iutA*), salmochelin (*iroBCDEN*), and the *sitABCD* genes (35, 53). The *sitABCD* genes were also chromosomally integrated. The other putative virulence genes found on pS88 were the increased serum survival gene *iss*, involved in complement resistance (17); the *etsABC* genes, encoding a putative type 1 secretion system (35); *ompT*, encoding an outer membrane protease (58); and *hlyF*, encoding a hemolysin (44). The S88 *ompT* gene is 100% homologous to the APEC O2 and O1 plasmid orthologs (34, 35) but differs significantly from the common *E. coli* chromosomal *ompT* gene. Therefore, we designate this gene *ompT*<sub>P</sub> (*ompT* of plasmids).

(iv) Comparison with other plasmids. BLAST comparisons of the overall pS88 sequence with other sequences revealed that pS88 is closely related to pAPEC-O2-ColV and pAPEC-O1-ColBM (Table 3). These plasmids (184,501 and 174,240 bp, respectively) came from avian pathogenic *E. coli* (APEC) O2:K1 and O1:K1 strains causing colibacillosis in chickens (34, 35). Alignment of the three plasmid sequences using the online comparison tool WebACT Artemis (1) is shown in Fig. 3. The line plot revealed several large blocks of highly homologous DNA between pS88 and the two APEC plasmids. Depending on the region examined, the genes and their organization were more similar to pAPEC-O1-ColBM or to pAPEC-O2-ColV. The virulence region, from the locus *iroBCDEN* to the locus *sitABCD*, appears to be highly homologous to the virulence region of pAPEC-O2-ColV (the “conserved” virulence region
<table><caption>TABLE 3—Continued</caption><thead><tr><th colspan="2">Position</th><th rowspan="2">ORF</th><th rowspan="2">Product</th><th colspan="2">pAPEC-O2-ColV homolog</th><th colspan="2">pAPEC-O1-ColBM homolog</th></tr><tr><th>Beginning</th><th>End</th><th>Name</th><th>Identity (%)</th><th>Name</th><th>Identity (%)</th></tr></thead><tbody><tr><td>110582</td><td>111217</td><td></td><td>Conserved hypothetical protein</td><td>O2ColV51</td><td>95.6</td><td>O1CoBM143</td><td>100</td></tr><tr><td>111327</td><td>111632</td><td></td><td>Conserved hypothetical protein</td><td>O2ColV50</td><td>99</td><td>O1CoBM144</td><td>100</td></tr><tr><td>112196</td><td>112378</td><td>iss</td><td>Hypothetical protein</td><td>O2ColV49.2</td><td>100</td><td>O1CoBM146</td><td>100</td></tr><tr><td>112492</td><td>112785</td><td>yqfG</td><td>Iss (Increased serum survival)</td><td>O2ColV49</td><td>100</td><td>O1CoBM126</td><td>100</td></tr><tr><td>113412</td><td>113777</td><td></td><td>Transposase ORF A, IS2</td><td>O2ColV48.2</td><td>99.6</td><td>O1CoBM149</td><td>100</td></tr><tr><td>113735</td><td>114640</td><td>yqfG</td><td>Transposase ORF 2, IS2</td><td>O2ColV48</td><td>97.3</td><td>O1CoBM150</td><td>100</td></tr><tr><td>115306</td><td>115449</td><td></td><td>Hypothetical protein</td><td></td><td></td><td>O1CoBM151</td><td>99.75</td></tr><tr><td>115864</td><td>117057</td><td></td><td>Conserved hypothetical protein; putative GTPase</td><td>O2ColV47</td><td>100</td><td>O1CoBM152</td><td>100</td></tr><tr><td>117190</td><td>117507</td><td></td><td>Putative transposase ORF B (fragment), IS2</td><td>O2ColV46</td><td>100</td><td>O1CoBM153</td><td>100</td></tr><tr><td>117694</td><td>118560</td><td></td><td>Putative transposase (fragment)</td><td>O2ColV45</td><td>95.8</td><td></td><td></td></tr><tr><td>118538</td><td>118648</td><td></td><td>Putative transposase (fragment)</td><td>O2ColV45</td><td>83.9</td><td></td><td></td></tr><tr><td>118697</td><td>119083</td><td></td><td>Putative transposase (fragment)</td><td>O2ColV45</td><td>93</td><td></td><td></td></tr><tr><td>119120</td><td>119308</td><td></td><td>Conserved hypothetical protein</td><td></td><td></td><td>O1CoBM154</td><td>100</td></tr><tr><td>119318</td><td>120259</td><td>etsC</td><td>Conserved hypothetical protein</td><td></td><td></td><td>O1CoBM155</td><td>100</td></tr><tr><td>121056</td><td>122426</td><td>etsB</td><td>Putative type I secretion outer membrane protein EtsC</td><td>O2ColV35</td><td>100</td><td>O1CoBM156</td><td>100</td></tr><tr><td>122430</td><td>124370</td><td></td><td>Putative type I secretion ATP-binding protein EtsB</td><td>O2ColV34</td><td>99.9</td><td>O1CoBM199</td><td>100</td></tr><tr><td>124367</td><td>125575</td><td>etsA</td><td>Putative type I secretion membrane-fusion protein EtsA</td><td>O2ColV33</td><td>98.7</td><td>O1CoBM198</td><td>99.85</td></tr><tr><td>126448</td><td>126738</td><td></td><td>Putative transposase ORF B (fragment), IS3 family</td><td></td><td></td><td>O1CoBM197</td><td>99.75</td></tr><tr><td>126687</td><td>126860</td><td></td><td>Putative transposase ORF B (fragment), IS3 family</td><td></td><td></td><td>O1CoBM196</td><td>100</td></tr><tr><td>127008</td><td>127898</td><td></td><td>Putative transposase ORF B, IS3 family, IS57 group</td><td>O2ColV95</td><td>99.2</td><td>O1CoBM194</td><td>100</td></tr><tr><td>127895</td><td>128221</td><td></td><td>Transposase ORF A, IS3 family, IS37 group</td><td>O2ColV96</td><td>93.5</td><td>O1CoBM192</td><td>100</td></tr><tr><td>128405</td><td>128563</td><td></td><td>Putative transposase ORF B (fragment), IS family, IS407 group</td><td>O2ColV29</td><td>98.1</td><td>O1CoBM154</td><td>100</td></tr><tr><td>129343</td><td>129732</td><td></td><td>Hypothetical protein</td><td>O2ColV27</td><td>96.9</td><td>O1CoBM155</td><td>100</td></tr><tr><td>129836</td><td>130789</td><td>ompT<sub>P</sub></td><td>Outer membrane protease (omptin)</td><td>O2ColV26</td><td>100</td><td></td><td></td></tr><tr><td>131222</td><td>132331</td><td>hlyF</td><td>Hemolysin HlyF</td><td>O2ColV24</td><td>100</td><td>O1CoBM189</td><td>100</td></tr><tr><td>132394</td><td>133302</td><td></td><td>Conserved hypothetical protein; putative Mlg-14 protein</td><td></td><td></td><td>O1CoBM188</td><td>99.67</td></tr></tbody></table>

immune system (see below); they were transcribed in both directions and showed sharp deviations from the average G+C content (29.9% to 63.6%), suggesting a foreign mosaic origin. Thirty-five ORFs of unknown function and 14 insertion sequence-like genes were scattered throughout the plasmid. Two different colicins, colicin Ia and colicin V, along with their immunity protein, were encoded by pS88.

(ii) **Replication, transfer, and maintenance regions of pS88.** Plasmid pS88 harbors two replicons. The first replicon region, RepFIB, contains the typical replication gene *repA* and the site-specific integrase *int* (55). The second, RepFIIA, encodes the CopB repressor and the RepA1 replication proteins. Thus, pS88 appears to belong to both the IncFI and IncFII incompatibility groups. The complete F-like transfer region of pS88 spans 31,485 bp and contains 32 genes (from *traM* to *finO*). The DNA sequences of the three regulator genes *traM*, *traJ*, and *traY*, relative to publicly available databases, appear to be characteristic of an F-type plasmid rather than an R1-type plasmid (15) (Fig. 2). None of the other *tra* genes were discriminated (as exemplified by *traT*), except for *traS*, which was closer to that of R1-type plasmids (Fig. 2). Upstream of the transfer locus lie genes involved in single-stranded DNA transfer; *ssb*, the gene encoding single-stranded binding protein; and *psiA* and *psiB*, plasmid SOS inhibition genes (6). Two loci putatively involved in plasmid maintenance were identified. One, close to the transfer region, was composed of the *sopA* and *sopB* genes, coding for the plasmid partition proteins (45). Located downstream of the RepFIIA replicon, the *yacABC* operon may represent a toxin-antitoxin plasmid stability system of which a second copy, albeit truncated, lies between the colicin V and salmochelin operons.

(iii) **Putative virulence region of S88.** The putative virulence region of pS88 harbored three different iron uptake systems, namely, aerobactin (*iucABCD* and *iutA*), salmochelin (*iroBCDEN*), and the *sitABCD* genes (35, 53). The *sitABCD* genes were also chromosomally integrated. The other putative virulence genes found on pS88 were the increased serum survival gene *iss*, involved in complement resistance (17); the *etsABC* genes, encoding a putative type 1 secretion system (35); *ompT*, encoding an outer membrane protease (58); and *hlyF*, encoding a hemolysin (44). The S88 *ompT* gene is 100% homologous to the APEC O2 and O1 plasmid orthologs (34, 35) but differs significantly from the common *E. coli* chromosomal *ompT* gene. Therefore, we designate this gene *ompT*<sub>P</sub> (*ompT* of plasmids).

(iv) **Comparison with other plasmids.** BLAST comparisons of the overall pS88 sequence with other sequences revealed that pS88 is closely related to pAPEC-O2-ColV and pAPEC-O1-ColBM (Table 3). These plasmids (184,501 and 174,240 bp, respectively) came from avian pathogenic *E. coli* (APEC) O2:K1 and O1:K1 strains causing colibacillosis in chickens (34, 35). Alignment of the three plasmid sequences using the online comparison tool WebACT Artemis (1) is shown in Fig. 3. The line plot revealed several large blocks of highly homologous DNA between pS88 and the two APEC plasmids. Depending on the region examined, the genes and their organization were more similar to pAPEC-O1-ColBM or to pAPEC-O2-ColV. The virulence region, from the locus *iroBCDEN* to the locus *sitABCD*, appears to be highly homologous to the virulence region of pAPEC-O2-ColV (the “conserved” virulence region
<figure><img src="image_3.png" /><figcaption>FIG. 2. Phylogenetic trees generated by the neighbor-joining method for *traJ*, *traM*, *traS*, *traT*, and *traY* sequences of several reference *E. coli* conjugative plasmids. Numbers at the branches are bootstrap proportions (displayed if >75) obtained from 100 replicates. Sequences were extracted from GenBank, except for plasmid pS88: pAPEC-O2-ColV (strain APEC O2; accession number AY545598), pAPEC-O1-ColBM (strain APEC O1; accession number DQ381420), plasmid F (accession number AP001918), p1658-97 (accession number AF550679), p53638 (accession number CP001064), pMAR7 (accession number DQ388534), pRK100 (accession number AY230886), pSMS-3-5 (accession number CP000971), pUTI89 (accession number CP000244), pVM01 (accession number EU330199), and plasmid R1 (accession number X13681) and its derepressed mutant pR1-19 (accession number M19710). Plasmid pS88 and the two APEC plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM are highlighted in bold.</figcaption></figure>

described by Johnson et al. [35]) and to have a highly similar
organization (Fig. 3). In contrast, a block of DNA containing
the loci *etsABC*, *hlyF*, and *ompT*, central to the virulence
region of pS88, was inverted and relocated to the end of the
virulence region in pAPEC-O1-ColBM. As expected, the
transfer loci were highly homologous, but in pS88 the three
transfer regulator genes *traM*, *traJ*, and *traY* (at the beginning
of the *tra* operon) had a higher percent identity to those of
pAPEC-O1-ColBM than to those of pAPEC-O2-ColV (100%
versus 85%, 99% versus 25%, and 99% versus 53%, respec-
tively). The contrary was observed for *traS* (Fig. 2). Between
the locus *tra* and the virulence region in pS88 lies a small
region containing the colicin Ia and V operons and the par-
tially duplicated *yacABC* operon. This contrasts with the pres-
ence at this location of a larger region in both APEC plasmids,
harboring putative virulence loci such as *eit* and *tsh* (the “vari-
able” virulence region described by Johnson et al. [35]). How-
ever, these “variable” virulence regions contain the complete
ColV operon in pAPEC-O2-ColV, as in pS88, while it is trun-
cated in pAPEC-O1-ColBM. Finally, the maintenance region
downstream of the virulence region appears to be more closely
related to the corresponding region of pAPEC-O1-ColBM
than to that of pAPEC-O2-ColV. For example, the partition
system present in pAPEC-O2-ColV (*parAB*) differs from that
of pAPEC-O1-ColBM and pS88 (*sopAB*).

**Molecular epidemiology of the plasmid.** The distribution of 11 ORFs or genes scattered throughout pS88 in a representative collection of worldwide human meningitis strains and in several nonmeningitis *E. coli* strains genetically related to S88 is shown in Table 1. Strains positive for all 11 screened ORFs were also screened for *eitB* and *tsh* genes, representing the “variable” virulence region. Strains harboring all 11 pS88 genes and lacking the “variable” virulence region were considered to harbor a pS88-like plasmid. Among human meningitis strains, a pS88-like plasmid was frequently observed in the highly virulent subgroup B2₁ defined by a specific ST (ST29<sub>Whittam</sub> [www.shigatox.net], ST95<sub>Achtmann</sub> [www.mlst.net], or B2-IX of De-namur's scheme) (10, 40). Indeed, a pS88-like plasmid was found in all the ST29<sup>O45</sup> strains and about half the ST29<sup>O18</sup> and ST29<sup>O1</sup> strains. Outside of this subgroup ST29/B2₁, a pS88-like plasmid was observed in a few (2/11) O83:K1 strains (ST697<sup>O83</sup> and ST692<sup>O83</sup>) and never in other major-sequence O-type strains (ST304<sup>O16</sup>, ST301<sup>O7</sup>, and ST100<sup>O1</sup>). As this plasmid appears to be highly frequent in group B2 and especially in subgroup ST29/B2₁, we investigated several nonmeningitis human and nonhuman strains belonging to the ST29/B2₁ subgroup. Interestingly, the four ST29<sup>O45</sup> APEC strains from Spain also harbored a pS88-like plasmid. Among the ST29<sup>O2</sup> strains, the pS88-like plasmid was found in several human strains, including strain ECOR62 but not strain
<figure><img src="image_4.png" /><figcaption>FIG. 2. Phylogenetic trees generated by the neighbor-joining method for *traJ*, *traM*, *traS*, *traT*, and *traY* sequences of several reference *E. coli* conjugative plasmids. Numbers at the branches are bootstrap proportions (displayed if >75) obtained from 100 replicates. Sequences were extracted from GenBank, except for plasmid pS88: pAPEC-O2-ColV (strain APEC O2; accession number AY545598), pAPEC-O1-ColBM (strain APEC O1; accession number DQ381420), plasmid F (accession number AP001918), p1658-97 (accession number AF550679), p53638 (accession number CP001064), pMAR7 (accession number DQ388534), pRK100 (accession number AY230886), pSMS-3-5 (accession number CP000971), pUTI89 (accession number CP000244), pVM01 (accession number EU330199), and plasmid R1 (accession number X13681) and its derepressed mutant pR1-19 (accession number M19710). Plasmid pS88 and the two APEC plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM are highlighted in bold.</figcaption></figure>

described by Johnson et al. [35]) and to have a highly similar
organization (Fig. 3). In contrast, a block of DNA containing
the loci *etsABC*, *hlyF*, and *ompT*, central to the virulence
region of pS88, was inverted and relocated to the end of the
virulence region in pAPEC-O1-ColBM. As expected, the
transfer loci were highly homologous, but in pS88 the three
transfer regulator genes *traM*, *traJ*, and *traY* (at the beginning
of the *tra* operon) had a higher percent identity to those of
pAPEC-O1-ColBM than to those of pAPEC-O2-ColV (100%
versus 85%, 99% versus 25%, and 99% versus 53%, respec-
tively). The contrary was observed for *traS* (Fig. 2). Between
the locus *tra* and the virulence region in pS88 lies a small
region containing the colicin Ia and V operons and the par-
tially duplicated *yacABC* operon. This contrasts with the pres-
ence at this location of a larger region in both APEC plasmids,
harboring putative virulence loci such as *eit* and *tsh* (the “vari-
able” virulence region described by Johnson et al. [35]). How-
ever, these “variable” virulence regions contain the complete
ColV operon in pAPEC-O2-ColV, as in pS88, while it is trun-
cated in pAPEC-O1-ColBM. Finally, the maintenance region
downstream of the virulence region appears to be more closely
related to the corresponding region of pAPEC-O1-ColBM
than to that of pAPEC-O2-ColV. For example, the partition
system present in pAPEC-O2-ColV (*parAB*) differs from that
of pAPEC-O1-ColBM and pS88 (*sopAB*).

**Molecular epidemiology of the plasmid.** The distribution of 11 ORFs or genes scattered throughout pS88 in a representative collection of worldwide human meningitis strains and in several nonmeningitis *E. coli* strains genetically related to S88 is shown in Table 1. Strains positive for all 11 screened ORFs were also screened for *eitB* and *tsh* genes, representing the “variable” virulence region. Strains harboring all 11 pS88 genes and lacking the “variable” virulence region were considered to harbor a pS88-like plasmid. Among human meningitis strains, a pS88-like plasmid was frequently observed in the highly virulent subgroup B2₁ defined by a specific ST (ST29<sub>Whittam</sub> [www.shigatox.net], ST95<sub>Achtmann</sub> [www.mlst.net], or B2-IX of De-namur's scheme) (10, 40). Indeed, a pS88-like plasmid was found in all the ST29<sup>O45</sup> strains and about half the ST29<sup>O18</sup> and ST29<sup>O1</sup> strains. Outside of this subgroup ST29/B2₁, a pS88-like plasmid was observed in a few (2/11) O83:K1 strains (ST697<sup>O83</sup> and ST692<sup>O83</sup>) and never in other major-sequence O-type strains (ST304<sup>O16</sup>, ST301<sup>O7</sup>, and ST100<sup>O1</sup>). As this plasmid appears to be highly frequent in group B2 and especially in subgroup ST29/B2₁, we investigated several nonmeningitis human and nonhuman strains belonging to the ST29/B2₁ subgroup. Interestingly, the four ST29<sup>O45</sup> APEC strains from Spain also harbored a pS88-like plasmid. Among the ST29<sup>O2</sup> strains, the pS88-like plasmid was found in several human strains, including strain ECOR62 but not strain
<figure><img src="image_4.png" /><figcaption>FIG. 3. Comparison of plasmid pS88 (133,853 bp) with plasmids pAPEC-O1-ColBM (174,241 bp) and pAPEC-O2-ColV (184,501 bp) using the line-plot representation of homologous regions. For convenience, the *traM* start codon was chosen as the beginning of the three sequences. Strand conservations are indicated in blue and strand inversions in red. Genes displayed are categorized using the color scheme described in Fig. 1. Variable and conserved virulence regions are defined as previously described by Johnson et al. (35).</figcaption></figure>

ECOR61; the two ST29O2 APEC strains harbored a pS88-close plasmid that was PCR negative for *cia* and *traJ*F*-plasmid, suggesting the presence of a pAPEC-O2-ColV-like plasmid.

PFGE of undigested DNAs of several strains representative of different sequence O types positive for the 11 pS88 genes showed that all harbored a plasmid of about 130 kb hybridizing with the *iroN* probe (Fig. 4).

Overall, our molecular epidemiology studies indicate that the pS88-like plasmid is not restricted to France, as such plasmids can be found in strains from the United States, Spain, or Sweden, and not only to meningitis strains (Table 1).

**Functional analysis. (i) Plasmid curation and experimental virulence.** Exposure to sodium dodecyl sulfate yielded S88 variants at a high rate (99%). One of these variants (CH7) was markedly less virulent than the wild-type strain in the animal model, with a level of bacteremia at least 2 log CFU/ml lower in two separate experiments (Table 4). To confirm that this was due to the loss of pS88, we complemented the S88 variant with pS88cat, using a double-conjugal plasmid transfer method. Virulence was fully restored with the complemented strain, designated S88E (Table 4). Comparative experiments with growth in serum showed that S88 and S88E were more resistant to serum bactericidal activity than CH7, with a mean difference of 1.1 log CFU/ml (*P* < 0.05) after 5 hours of incubation (data not shown).

**(ii) Mutagenesis.** As we have previously shown that *iroN* has a role in the virulence of strain C5 (O18:K1:H7) (47), we

<figure><img src="image_5.png" /><figcaption>FIG. 4. (A) PFGE of undigested DNAs from seven representative strains harboring the 11 ORFs characteristics of pS88. Lane 1, S145; lane 2, Ben1068; lane 3, S4; lane 4, S133; lane 5, S94; lane 6, HN30; lane 7, ECOR62; lane M, molecular size marker (50-kb DNA ladder). (B) Hybridization with *iroN*-specific probe on a Southern blot.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>FIG. 3. Comparison of plasmid pS88 (133,853 bp) with plasmids pAPEC-O1-ColBM (174,241 bp) and pAPEC-O2-ColV (184,501 bp) using the line-plot representation of homologous regions. For convenience, the *traM* start codon was chosen as the beginning of the three sequences. Strand conservations are indicated in blue and strand inversions in red. Genes displayed are categorized using the color scheme described in Fig. 1. Variable and conserved virulence regions are defined as previously described by Johnson et al. (35).</figcaption></figure>

ECOR61; the two ST29O2 APEC strains harbored a pS88-close plasmid that was PCR negative for *cia* and *traJ*F*-plasmid, suggesting the presence of a pAPEC-O2-ColV-like plasmid.

PFGE of undigested DNAs of several strains representative of different sequence O types positive for the 11 pS88 genes showed that all harbored a plasmid of about 130 kb hybridizing with the *iroN* probe (Fig. 4).

Overall, our molecular epidemiology studies indicate that the pS88-like plasmid is not restricted to France, as such plasmids can be found in strains from the United States, Spain, or Sweden, and not only to meningitis strains (Table 1).

**Functional analysis. (i) Plasmid curation and experimental virulence.** Exposure to sodium dodecyl sulfate yielded S88 variants at a high rate (99%). One of these variants (CH7) was markedly less virulent than the wild-type strain in the animal model, with a level of bacteremia at least 2 log CFU/ml lower in two separate experiments (Table 4). To confirm that this was due to the loss of pS88, we complemented the S88 variant with pS88cat, using a double-conjugal plasmid transfer method. Virulence was fully restored with the complemented strain, designated S88E (Table 4). Comparative experiments with growth in serum showed that S88 and S88E were more resistant to serum bactericidal activity than CH7, with a mean difference of 1.1 log CFU/ml (*P* < 0.05) after 5 hours of incubation (data not shown).

**(ii) Mutagenesis.** As we have previously shown that *iroN* has a role in the virulence of strain C5 (O18:K1:H7) (47), we

<figure><img src="image_6.png" /><figcaption>FIG. 4. (A) PFGE of undigested DNAs from seven representative strains harboring the 11 ORFs characteristics of pS88. Lane 1, S145; lane 2, Ben1068; lane 3, S4; lane 4, S133; lane 5, S94; lane 6, HN30; lane 7, ECOR62; lane M, molecular size marker (50-kb DNA ladder). (B) Hybridization with *iroN*-specific probe on a Southern blot.</figcaption></figure>
<table><caption>TABLE 4. Mean bacteremia values in a neonatal rat model challenged with S88 and its mutants, variants, and transconjugants</caption><thead><tr><th>Expt and strain</th><th>Strain description</th><th>No. of infected animals</th><th>Mean level of bacteremia, log CFU/ml (SD)</th></tr></thead><tbody><tr><td rowspan="2">1</td><td>S88</td><td>S88 wild type</td><td>10</td><td>6.02 (1.52)</td></tr><tr><td>CH7</td><td>S88Δp</td><td>10</td><td>3.54 (0.71)<sup>a</sup></td></tr><tr><td rowspan="2">2</td><td>S88</td><td>S88 wild type</td><td>10</td><td>5.26 (1.9)</td></tr><tr><td>S88E</td><td>S88Δp (pS88cat)</td><td>10</td><td>5.95 (1.65)</td></tr><tr><td rowspan="2">3</td><td>S88</td><td>S88 wild type</td><td>13</td><td>6.50 (1.67)</td></tr><tr><td>CH1</td><td>S88 (iroN::cat)</td><td>13</td><td>5.67 (1.87)<sup>b</sup></td></tr><tr><td rowspan="2">4</td><td>S88</td><td>S88 wild type</td><td>9</td><td>6.17 (0.81)</td></tr><tr><td>CH23</td><td>S88 (iut::cat)</td><td>9</td><td>5.85 (1.56)</td></tr><tr><td rowspan="2">5</td><td>S88</td><td>S88 wild type</td><td>14</td><td>5.71 (1.20)</td></tr><tr><td>CH19</td><td>S88 (sit::cat)</td><td>14</td><td>5.54 (1.35)</td></tr></tbody></table>

examined its involvement, as well as that of the other two iron uptake systems (aerobactin and the *sit* operon), in the virulence linked to pS88. Only *sitABCD* was also present on the chromosome. A CH1 mutant with the salmochelin receptor gene (*iroN*) disrupted was slightly less virulent than the wild-type strain, with a decrease in bacteremia of 0.9 log CFU/ml (Table 4). Two other mutants were also constructed: CH19, which lacks both the chromosome- and plasmid-encoded copies of the *sit* operon (double deletions were obtained in one step of mutagenesis), and CH23, which lacks the aerobactin receptor. The virulence of these two mutants was similar to that of the wild-type strain (Table 4).

DISCUSSION

S88, the representative strain of the recently described French meningitis clone O45:K1:H7, harbors only a few of the specific virulence factors, such as the K1 capsule antigen and salmochelin, known to influence the onset of meningitis in newborns (13). The *E. coli* genomic project ColiScope, which include the sequencing of S88, provides the opportunity to discover new genetic determinants involved in the virulence of this clone. Indeed, we recently demonstrated the major role of the O-antigen gene cluster, which, surprisingly, encodes a new O antigen slightly different from the O45 reference antigen (50). Sequencing of S88 revealed the presence of a large plasmid encoding two bacteriocins: colicin V and colicin Ia. Although ColV plasmids have long been known to contribute to the virulence of ExPEC strains (3, 4, 54, 57, 64, 66), pS88 is the first ColV plasmid of a human ExPEC strain to be entirely sequenced. However, ColV plasmids have also been linked to APEC virulence, and recently an APEC ColV plasmid (pAPEC-O2-ColV) and a related plasmid (pAPEC-O1-ColBM) have been sequenced (34, 35).

Comparison of pS88 with other sequenced plasmids re-

vealed strong homologies with pAPEC-O2-ColV and pAPEC-O1-ColBM. One of the most striking homologies involves a “conserved” virulence region in APEC plasmids. This region contains numerous known or putative virulence determinants (*iroBCDEN*, *iss*, *etsABC*, *ompT*<sub>P</sub>, *hlyF*, *iucABCD/iutA*, and *sitABCD*) but also ORFs of unknown function, which are all present in the three plasmids in a DNA block less than 60 kb long. Several of these virulence genes have been described as chromosomally encoded in pathogenicity islands, including the salmochelin locus in PAI III<sub>536</sub> (16), the aerobactin locus in PAI I<sub>CFT073</sub> (65), and the *sitABCD* locus in *Salmonella* pathogenicity island 1 (31), or on phage-related DNA, such as *iss* in several wholly sequenced uropathogenic *E. coli* (UPEC) genomes (37). In contrast, the three contiguous putative virulence determinants *etsABC*, *ompT*<sub>P</sub>, and *hlyF* have never been described on the chromosomes of *E. coli* or related species, including those examined for the ColiScope project. These genes may therefore be plasmid specific and represent a signature for the presence of a virulence plasmid in a given strain. The absence of these three genes in *E. coli* chromosomes suggests that they were acquired more recently than other virulence traits that have been integrated in either the chromosome or the plasmid. Their marked %GC differences (48%, 43%, and 38%, respectively) indicate that this nucleus was constructed step by step rather than being acquired “en bloc.” This is supported by the fact that *hlyF* can be found alone in plasmid p1658-97 (accession number AF550679) and associated with *ompT*<sub>P* (but without *ets*) in plasmid pSMS-3-5 (accession number CP000971).

The other region showing strong DNA homology with APEC plasmids was, as expected, the transfer region. However, several interesting features were noted. *traY*, one of the three regulatory genes of the *tra* locus, was used initially by Boyd, and since by several other authors, to discriminate between F- and R1-type plasmids (15, 32). Phylogenetic relatedness inferred from publicly available DNA sequences of *traY* and the two adjacent genes, *traJ* and *traM*, clearly show that pS88 may be considered an F-plasmid, like pAPEC-O1-ColBM, while pAPEC-O2-ColV is closer to an R1-type plasmid. None of the other genes, except for *traS*, could discriminate, owing to their strong homology (*traT* for instance). Interestingly, *traS* was paradoxically closer to that of pAPEC-O2-ColV than to that of pAPEC-O1-ColBM. This supports the mosaic structure of the pS88 transfer locus, in keeping with the results of previous *tra* sequence analyses (15). In contrast to genetic and biochemical mechanisms, we know little of the environmental and physiological factors that affect conjugal transfer, even though they are likely to play an important role (23). The striking segregation of the three regulatory genes, and especially *traJ*, into two groups may correspond to two different regulatory systems adapted to two different environments or two different lifestyles. Of note, *traJ* of the R1-like plasmid is known to have a role in the virulence of the major neonatal meningitis clone O18:K1:H7 (represented by strain UTI89 in Fig. 2) (5). In this clone, *TraJ* contributes significantly to the survival of *E. coli* K1 inside professional phagocytes (27). Therefore, it would be of interest to determine whether the distantly related pS88 *traJ* gene (related to an F-like plasmid) has a similar or a different role.

One particular feature of pS88 is the cooccurrence of the

<footnote><sup>a</sup> *P* < 0.05 versus S88 bacterial count in experiment 1.</footnote>

<footnote><sup>b</sup> *P* < 0.05 versus S88 bacterial count in experiment 3.</footnote>
<table><caption>TABLE 4. Mean bacteremia values in a neonatal rat model challenged with S88 and its mutants, variants, and transconjugants</caption><thead><tr><th>Expt and strain</th><th>Strain description</th><th>No. of infected animals</th><th>Mean level of bacteremia, log CFU/ml (SD)</th></tr></thead><tbody><tr><td rowspan="2">1</td><td>S88</td><td>S88 wild type</td><td>10</td><td>6.02 (1.52)</td></tr><tr><td>CH7</td><td>S88Δp</td><td>10</td><td>3.54 (0.71)<sup>a</sup></td></tr><tr><td rowspan="2">2</td><td>S88</td><td>S88 wild type</td><td>10</td><td>5.26 (1.9)</td></tr><tr><td>S88E</td><td>S88Δp (pS88cat)</td><td>10</td><td>5.95 (1.65)</td></tr><tr><td rowspan="2">3</td><td>S88</td><td>S88 wild type</td><td>13</td><td>6.50 (1.67)</td></tr><tr><td>CH1</td><td>S88 (iroN::cat)</td><td>13</td><td>5.67 (1.87)<sup>b</sup></td></tr><tr><td rowspan="2">4</td><td>S88</td><td>S88 wild type</td><td>9</td><td>6.17 (0.81)</td></tr><tr><td>CH23</td><td>S88 (iut::cat)</td><td>9</td><td>5.85 (1.56)</td></tr><tr><td rowspan="2">5</td><td>S88</td><td>S88 wild type</td><td>14</td><td>5.71 (1.20)</td></tr><tr><td>CH19</td><td>S88 (sit::cat)</td><td>14</td><td>5.54 (1.35)</td></tr></tbody></table>

examined its involvement, as well as that of the other two iron uptake systems (aerobactin and the *sit* operon), in the virulence linked to pS88. Only *sitABCD* was also present on the chromosome. A CH1 mutant with the salmochelin receptor gene (*iroN*) disrupted was slightly less virulent than the wild-type strain, with a decrease in bacteremia of 0.9 log CFU/ml (Table 4). Two other mutants were also constructed: CH19, which lacks both the chromosome- and plasmid-encoded copies of the *sit* operon (double deletions were obtained in one step of mutagenesis), and CH23, which lacks the aerobactin receptor. The virulence of these two mutants was similar to that of the wild-type strain (Table 4).

DISCUSSION

S88, the representative strain of the recently described French meningitis clone O45:K1:H7, harbors only a few of the specific virulence factors, such as the K1 capsule antigen and salmochelin, known to influence the onset of meningitis in newborns (13). The *E. coli* genomic project ColiScope, which include the sequencing of S88, provides the opportunity to discover new genetic determinants involved in the virulence of this clone. Indeed, we recently demonstrated the major role of the O-antigen gene cluster, which, surprisingly, encodes a new O antigen slightly different from the O45 reference antigen (50). Sequencing of S88 revealed the presence of a large plasmid encoding two bacteriocins: colicin V and colicin Ia. Although ColV plasmids have long been known to contribute to the virulence of ExPEC strains (3, 4, 54, 57, 64, 66), pS88 is the first ColV plasmid of a human ExPEC strain to be entirely sequenced. However, ColV plasmids have also been linked to APEC virulence, and recently an APEC ColV plasmid (pAPEC-O2-ColV) and a related plasmid (pAPEC-O1-ColBM) have been sequenced (34, 35).

Comparison of pS88 with other sequenced plasmids re-

vealed strong homologies with pAPEC-O2-ColV and pAPEC-O1-ColBM. One of the most striking homologies involves a “conserved” virulence region in APEC plasmids. This region contains numerous known or putative virulence determinants (*iroBCDEN*, *iss*, *etsABC*, *ompT*, *hlyF*, *iucABCD/iutA*, and *sitABCD*) but also ORFs of unknown function, which are all present in the three plasmids in a DNA block less than 60 kb long. Several of these virulence genes have been described as chromosomally encoded in pathogenicity islands, including the salmochelin locus in PAI III<sub>536</sub> (16), the aerobactin locus in PAI I<sub>CFT073</sub> (65), and the *sitABCD* locus in *Salmonella* pathogenicity island 1 (31), or on phage-related DNA, such as *iss* in several wholly sequenced uropathogenic *E. coli* (UPEC) genomes (37). In contrast, the three contiguous putative virulence determinants *etsABC*, *ompT*, and *hlyF* have never been described on the chromosomes of *E. coli* or related species, including those examined for the ColiScope project. These genes may therefore be plasmid specific and represent a signature for the presence of a virulence plasmid in a given strain. The absence of these three genes in *E. coli* chromosomes suggests that they were acquired more recently than other virulence traits that have been integrated in either the chromosome or the plasmid. Their marked %GC differences (48%, 43%, and 38%, respectively) indicate that this nucleus was constructed step by step rather than being acquired “en bloc.” This is supported by the fact that *hlyF* can be found alone in plasmid p1658-97 (accession number AF550679) and associated with *ompT* (but without *ets*) in plasmid pSMS-3-5 (accession number CP000971).

The other region showing strong DNA homology with APEC plasmids was, as expected, the transfer region. However, several interesting features were noted. *traY*, one of the three regulatory genes of the *tra* locus, was used initially by Boyd, and since by several other authors, to discriminate between F- and R1-type plasmids (15, 32). Phylogenetic relatedness inferred from publicly available DNA sequences of *traY* and the two adjacent genes, *traJ* and *traM*, clearly show that pS88 may be considered an F-plasmid, like pAPEC-O1-ColBM, while pAPEC-O2-ColV is closer to an R1-type plasmid. None of the other genes, except for *traS*, could discriminate, owing to their strong homology (*traT* for instance). Interestingly, *traS* was paradoxically closer to that of pAPEC-O2-ColV than to that of pAPEC-O1-ColBM. This supports the mosaic structure of the pS88 transfer locus, in keeping with the results of previous *tra* sequence analyses (15). In contrast to genetic and biochemical mechanisms, we know little of the environmental and physiological factors that affect conjugal transfer, even though they are likely to play an important role (23). The striking segregation of the three regulatory genes, and especially *traJ*, into two groups may correspond to two different regulatory systems adapted to two different environments or two different lifestyles. Of note, *traJ* of the R1-like plasmid is known to have a role in the virulence of the major neonatal meningitis clone O18:K1:H7 (represented by strain UTI89 in Fig. 2) (5). In this clone, *TraJ* contributes significantly to the survival of *E. coli* K1 inside professional phagocytes (27). Therefore, it would be of interest to determine whether the distantly related pS88 *traJ* gene (related to an F-like plasmid) has a similar or a different role.

One particular feature of pS88 is the cooccurrence of the

<footnote><sup>a</sup> *P* < 0.05 versus S88 bacterial count in experiment 1.</footnote>

<footnote><sup>b</sup> *P* < 0.05 versus S88 bacterial count in experiment 3.</footnote>
two bacteriocins ColV and ColIa. Cells producing two bacteriocins can kill single colicin producers and have fitness advantages, as bacteriocin can be produced under different conditions of stress (25). In a collection of 1,308 animal and human *E. coli* strains, Jeziorowski and Gordon showed that colicin Ia and colicin V were both present in a given strain more often than would be expected by chance and that the two bacteriocin genes were carried on a single plasmid (32). Based on sequence polymorphism analysis of the two operons and their rate of occurrence, it has been postulated that this cooccurrence may result from the bacteriocin V operon moving onto colicin Ia plasmids (32). The first complete sequence of a plasmid harboring both bacteriocins throws light on the coevolution of the two bacteriocins. First, we found that the two bacteriocin loci were located close together. This would facilitate their horizontal cotransfer on plasmid DNA segments. Although we observed no mobile genetic elements flanking the ColV and ColIa loci, the locus *yacABC* upstream of the ColIa-encoding genes was partially duplicated downstream of the ColV genes, possibly indicating a recombination event in which the two colicins were acquired in a single step. Therefore, pS88, which resembles pAPEC-O2-ColV and pAPEC-O1-ColBM, may have evolved by cotransfer of bacteriocins Ia and V with a recombination event between two conserved regions, the virulence region and the transfer region. This cotransfer might have provided the host strain with a fitness advantage and have favored the spread of this plasmid to different clonal groups.

Our molecular epidemiology studies of pS88 indicates that the pS88-like plasmid is not restricted to the virulent O45: K1:H7 clone but is also distributed, albeit less frequently, in other *E. coli* clonal groups such as O18:K1, O1:K1, O2:K1, and O83:K1. Intriguingly, these clones (except for the less extensively studied group O83:K1) may possess the same outer membrane protein profile, designed OMP9 (2, 48, 61, 67). OMP9 is linked to a particular protein (called PCP) encoded by a large IncFI plasmid that has not been further characterized (42). Moreover, OMP9 has never been described in clones O16:K1 and O7:K1, in which we found no pS88-like plasmid. This strongly suggests that pS88 harbors the genes encoding the PCP. We also found that all B2₁ O45:K1:H7 strains, both avian and human, harbor a pS88-like plasmid, further supporting the genetic relatedness of avian and human O45:K1:H7 strains (10). This is consistent with other observations suggesting that avian species may be the source of neonatal meningitis strains (22, 36, 46). The monomorphism of the plasmid content observed here in B2₁ O45:K1 strains and of the chromosomal determinants previously observed in these strains (13) suggests recent clonal emergence. Moreover the high prevalence of pS88-like plasmids among ST29/B21 strains (40/56, 71%) compared to other STs could represent an example of fine-tuning between a plasmid and a particular genetic background. Finally, the role of pS88 does not appear be restricted to meningeal virulence, as some members of the O2:K1 clonal group, known to cause urosepsis but not meningitis (10), also harbor a pS88-like plasmid (Table 1). Thus, the virulence mechanisms of this plasmid may have implications that go beyond a particular clone or pathotype.

To determine whether plasmid pS88 is directly involved in the ability of strain S88 to induce high-level bacteremia, the

main step in the pathogenesis of neonatal meningitis, we obtained an S88 variant lacking pS88. The variant produced a far lower level of bacteremia in the rat model (at least 2 log units lower). In order to confirm that the fall in virulence was due to loss of pS88, we reinserted a tagged pS88 in the cured strain S88. The resulting variant was as virulent as the wild-type strain. These results demonstrate the key role of the pS88 plasmid in the virulence of strain S88. Although numerous studies have established a link between the ColV plasmid and the virulence of various avian and human ExPEC strains (56), this is the first direct evidence supporting a role of a ColV plasmid in a specific step of neonatal meningitis, namely, sustained high-level bacteremia. This property may be due in part to the contribution of pS88 in resistance to serum bactericidal activity that we observed.

In a first attempt to understand the mechanism by which the plasmid contributes to the virulence of S88, we examined the three iron uptake systems encoded by the plasmid, namely, the siderophores salmochelin and aerobactin and the Sit system. Iron is essential for bacterial growth, but its availability within the host is limited (26). We have previously shown that salmochelin is involved in the high-level bacteremia achieved by the representative O18:K1:H7 strain C5 (47), in which this factor is chromosome borne. Hence, we examined whether this siderophore was also involved in the virulence of strain S88. We also investigated the respective virulence roles of the other two plasmid-encoded iron uptake systems, namely, aerobactin and the Sit system. Aerobactin is the most frequent siderophore in neonatal meningitis *E. coli* (NMEC) strains, after yersiniabactin (18, 13). Although this iron uptake system has been implicated in virulence in several experimental models (21, 24, 43, 60) its role in neonatal meningitis remains to be determined. The *sitABCD* operon, encoding a member of the family of ATP-binding cassette (ABC) proteins initially described for *Salmonella enterica*, was first functionally characterized as a manganese and iron transporter in an APEC strain (53). This newly described system is required for full virulence of *Salmonella enterica* serovar Typhimurium and APEC (31, 52). However, little is known of its distribution in human ExPEC strains or its role in their virulence (36, 42, 52).

As previously reported with strain C5 (47), we found that the virulence of strain S88 was attenuated when *iroN* was lacking. In contrast, the absence of aerobactin and the Sit system had no discernible influence on the virulence of strain S88. Our results thus prove the key role of salmochelin in the bacteremic step of *E. coli* meningitis. However, the lower virulence of the *iro*-defective mutant (~1 log CFU/ml lower) cannot alone explain the marked loss of virulence observed with the variant lacking the whole plasmid (>2 log CFU/ml). The fact that the loss of the other two siderophores did not influence the level of bacteremia does not totally exclude a role in the virulence of S88, as the *iro* locus may compensate for their loss. Further studies with double and triple mutants are under way. Alternatively, these siderophores may be involved in steps other than sustained bacteremia, such as gut translocation or cerebrospinal fluid invasion. Other iron uptake systems such as yersiniabactin, enterobactin, and the *FecABCD* system exist on the S88 chromosome (www.genoscope.fr) and may be able to compensate for the loss of the plasmid-encoded systems. Finally, it is likely that the virulence of the ColV plasmid involves
two bacteriocins ColV and ColIa. Cells producing two bacteriocins can kill single colicin producers and have fitness advantages, as bacteriocin can be produced under different conditions of stress (25). In a collection of 1,308 animal and human *E. coli* strains, Jeziorowski and Gordon showed that colicin Ia and colicin V were both present in a given strain more often than would be expected by chance and that the two bacteriocin genes were carried on a single plasmid (32). Based on sequence polymorphism analysis of the two operons and their rate of occurrence, it has been postulated that this cooccurrence may result from the bacteriocin V operon moving onto colicin Ia plasmids (32). The first complete sequence of a plasmid harboring both bacteriocins throws light on the coevolution of the two bacteriocins. First, we found that the two bacteriocin loci were located close together. This would facilitate their horizontal cotransfer on plasmid DNA segments. Although we observed no mobile genetic elements flanking the ColV and ColIa loci, the locus *yacABC* upstream of the ColIa-encoding genes was partially duplicated downstream of the ColV genes, possibly indicating a recombination event in which the two colicins were acquired in a single step. Therefore, pS88, which resembles pAPEC-O2-ColV and pAPEC-O1-ColBM, may have evolved by cotransfer of bacteriocins Ia and V with a recombination event between two conserved regions, the virulence region and the transfer region. This cotransfer might have provided the host strain with a fitness advantage and have favored the spread of this plasmid to different clonal groups.

Our molecular epidemiology studies of pS88 indicates that the pS88-like plasmid is not restricted to the virulent O45: K1:H7 clone but is also distributed, albeit less frequently, in other *E. coli* clonal groups such as O18:K1, O1:K1, O2:K1, and O83:K1. Intriguingly, these clones (except for the less extensively studied group O83:K1) may possess the same outer membrane protein profile, designed OMP9 (2, 48, 61, 67). OMP9 is linked to a particular protein (called PCP) encoded by a large IncFI plasmid that has not been further characterized (42). Moreover, OMP9 has never been described in clones O16:K1 and O7:K1, in which we found no pS88-like plasmid. This strongly suggests that pS88 harbors the genes encoding the PCP. We also found that all B2₁ O45:K1:H7 strains, both avian and human, harbor a pS88-like plasmid, further supporting the genetic relatedness of avian and human O45:K1:H7 strains (10). This is consistent with other observations suggesting that avian species may be the source of neonatal meningitis strains (22, 36, 46). The monomorphism of the plasmid content observed here in B2₁ O45:K1 strains and of the chromosomal determinants previously observed in these strains (13) suggests recent clonal emergence. Moreover the high prevalence of pS88-like plasmids among ST29/B21 strains (40/56, 71%) compared to other STs could represent an example of fine-tuning between a plasmid and a particular genetic background. Finally, the role of pS88 does not appear be restricted to meningeal virulence, as some members of the O2:K1 clonal group, known to cause urosepsis but not meningitis (10), also harbor a pS88-like plasmid (Table 1). Thus, the virulence mechanisms of this plasmid may have implications that go beyond a particular clone or pathotype.

To determine whether plasmid pS88 is directly involved in the ability of strain S88 to induce high-level bacteremia, the

main step in the pathogenesis of neonatal meningitis, we obtained an S88 variant lacking pS88. The variant produced a far lower level of bacteremia in the rat model (at least 2 log units lower). In order to confirm that the fall in virulence was due to loss of pS88, we reinserted a tagged pS88 in the cured strain S88. The resulting variant was as virulent as the wild-type strain. These results demonstrate the key role of the pS88 plasmid in the virulence of strain S88. Although numerous studies have established a link between the ColV plasmid and the virulence of various avian and human ExPEC strains (56), this is the first direct evidence supporting a role of a ColV plasmid in a specific step of neonatal meningitis, namely, sustained high-level bacteremia. This property may be due in part to the contribution of pS88 in resistance to serum bactericidal activity that we observed.

In a first attempt to understand the mechanism by which the plasmid contributes to the virulence of S88, we examined the three iron uptake systems encoded by the plasmid, namely, the siderophores salmochelin and aerobactin and the Sit system. Iron is essential for bacterial growth, but its availability within the host is limited (26). We have previously shown that salmochelin is involved in the high-level bacteremia achieved by the representative O18:K1:H7 strain C5 (47), in which this factor is chromosome borne. Hence, we examined whether this siderophore was also involved in the virulence of strain S88. We also investigated the respective virulence roles of the other two plasmid-encoded iron uptake systems, namely, aerobactin and the Sit system. Aerobactin is the most frequent siderophore in neonatal meningitis *E. coli* (NMEC) strains, after yersiniabactin (18, 13). Although this iron uptake system has been implicated in virulence in several experimental models (21, 24, 43, 60) its role in neonatal meningitis remains to be determined. The *sitABCD* operon, encoding a member of the family of ATP-binding cassette (ABC) proteins initially described for *Salmonella enterica*, was first functionally characterized as a manganese and iron transporter in an APEC strain (53). This newly described system is required for full virulence of *Salmonella enterica* serovar Typhimurium and APEC (31, 52). However, little is known of its distribution in human ExPEC strains or its role in their virulence (36, 42, 52).

As previously reported with strain C5 (47), we found that the virulence of strain S88 was attenuated when *iroN* was lacking. In contrast, the absence of aerobactin and the Sit system had no discernible influence on the virulence of strain S88. Our results thus prove the key role of salmochelin in the bacteremic step of *E. coli* meningitis. However, the lower virulence of the *iro*-defective mutant (~1 log CFU/ml lower) cannot alone explain the marked loss of virulence observed with the variant lacking the whole plasmid (>2 log CFU/ml). The fact that the loss of the other two siderophores did not influence the level of bacteremia does not totally exclude a role in the virulence of S88, as the *iro* locus may compensate for their loss. Further studies with double and triple mutants are under way. Alternatively, these siderophores may be involved in steps other than sustained bacteremia, such as gut translocation or cerebrospinal fluid invasion. Other iron uptake systems such as yersiniabactin, enterobactin, and the *FecABCD* system exist on the S88 chromosome (www.genoscope.fr) and may be able to compensate for the loss of the plasmid-encoded systems. Finally, it is likely that the virulence of the ColV plasmid involves
other determinants. The pS88 plasmid carries two genes, *iss* and *traT*, which play a role in serum survival and resistance to macrophage phagocytosis, respectively. Several other pS88 coding sequences have no homologs and could also encode new virulence determinants.

In conclusion, pS88 shares a similar structure with avian virulence plasmids, pointing to a possible common ancestor. It is an essential meningeal virulence determinant comparable to the K1 capsule and O45<sub>S88</sub> somatic antigens and is not restricted to the French O45:K1:H7 clone. A comprehensive study of the molecular mechanisms responsible for its virulence may have important implications, possibly beyond the pathogenesis of neonatal meningitis.

REFERENCES

1. Abbott, J. C., D. M. Aanensen, K. Rutherford, S. Butcher, and B. G. Spratt. 2005. WebACT—an online companion for the Artemis Comparison Tool. Bioinformatics 21:3665–3666.

2. Achtman, M., A. Mercer, B. Kusecek, A. Pohl, M. Heuzenroeder, W. Aaronson, A. Sutton, and R. P. Silver. 1983. Six widespread bacterial clones among *Escherichia coli* K1 isolates. Infect. Immun. 39:315–335.

3. Aguero, M. E., and F. C. Cabello. 1983. Relative contribution of ColV plasmid and K1 antigen to the pathogenicity of *Escherichia coli*. Infect. Immun. 40:359–368.

4. Aguero, M. E., G. de la Fuente, E. Vivaldi, and F. Cabello. 1989. ColV increases the virulence of *Escherichia coli* K1 strains in animal models of neonatal meningitis and urinary infection. Med. Microbiol. Immunol. (Berlin) 178:211–216.

5. Badger, J. L., C. A. Wass, S. J. Weissman, and K. S. Kim. 2000. Application of signature-tagged mutagenesis for identification of *Escherichia coli* K1 genes that contribute to invasion of human brain microvascular endothelial cells. Infect. Immun. 68:5056–5061.

6. Bagdasarian, M., A. Bailone, M. M. Bagdasarian, P. A. Manning, R. Lurz, K. N. Timmis, and R. Devoret. 1986. An inhibitor of SOS induction, specified by a plasmid locus in *Escherichia coli*. Proc. Natl. Acad. Sci. USA 83:5723–5726.

7. Barbe, V., D. Vallenet, N. Fonknechten, A. Kreimeyer, S. Oztas, L. Labarre, S. Cruveiller, C. Robert, S. Duprat, P. Wincker, L. N. Ornston, J. Weissenbach, P. Marliere, G. N. Cohen, and C. Medigue. 2004. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 32:5766–5779.

8. Bedford, H., J. de Louvois, S. Halket, C. Peckham, R. Hurley, and D. Harvey. 2001. Meningitis in infancy in England and Wales: follow up at age 5 years. BMJ 323:533–536.

9. Bidet, P., F. Mahjoub-Messai, J. Blanco, J. Blanco, M. Dehem, Y. Aujard, E. Bingen, and S. Bonacorsi. 2007. Combined multilocus sequence typing and O serogrouping distinguishes *Escherichia coli* subtypes associated with infant urosepsis and/or meningitis. J. Infect. Dis. 196:297–303.

10. Bidet, P., A. Metais, F. Mahjoub-Messai, L. Durand, M. Dehem, Y. Aujard, E. Bingen, X. Nassif, and S. Bonacorsi. 2007. Detection and identification by PCR of a highly virulent phylogenetic subgroup among extraintestinal pathogenic *Escherichia coli* B2 strains. Appl. Environ. Microbiol. 73:2373–2377.

11. 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. 177:642–650.

12. Bonacorsi, S., and E. Bingen. 2005. Molecular epidemiology of *Escherichia coli* causing neonatal meningitis. Int. J. Med. Microbiol. 295:373–381.

13. Bonacorsi, S., O. Clermont, V. Houdouin, C. Cordevant, N. Brahimi, A. Marecat, C. Tinsley, X. Nassif, M. Lange, and E. Bingen. 2003. Molecular analysis and experimental virulence of French and North American *Escherichia coli* neonatal meningitis isolates; identification of new virulent clone. J. Infect. Dis. 187:1895–1906.

14. Bonacorsi, S., S. Lefevre, O. Clermont, V. Houdouin, A. Bourrillon, C. Loirat, Y. Aujard, and E. Bingen. 2005. *Escherichia coli* strains causing urinary tract infection in uncircumcised infants resemble urosepsis-like adult strains. J. Urol. 173:195–197.

15. Boyd, E. F., C. W. Hill, S. M. Rich, and D. L. Hartl. 1996. Mosaic structure of plasmids from natural populations of *Escherichia coli*. Genetics 143:1091–1100.

16. Brzuszkiewicz, E., H. Bruggemann, H. Liesegang, M. Emmerth, T. Olschlager, G. Nagy, K. Albermann, C. Wagner, C. Buchrieser, L. Emody, G. Gottschalk, J. Hacker, and U. Dobrindt. 2006. How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic *Escherichia coli* strains. Proc. Natl. Acad. Sci. USA 103:12879–12884.

17. Chuba, P. J., M. A. Leon, A. Banerjee, and S. Palchaudhuri. 1989. Cloning

and DNA sequence of plasmid determinant *iss*, coding for increased serum survival and surface exclusion, which has homology with lambda DNA. Mol. Gen. Genet. 216:287–292.

18. Clermont, O., S. Bonacorsi, and E. Bingen. 2001. The *Yersinia* high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of *Escherichia coli*. FEMS Microbiol. Lett. 196:153–157.

19. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in *Escherichia coli* K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640–6645.

20. de Louvois, J., S. Halket, and D. Harvey. 2005. Neonatal meningitis in England and Wales: sequelae at 5 years of age. Eur. J. Pediatr. 164:730–734.

21. Dozois, C. M., F. Daigle, and R. Curtiss, 3rd. 2003. Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic *Escherichia coli* strain. Proc. Natl. Acad. Sci. USA 100:247–252.

22. Ewers, C., G. Li, H. Wilking, S. Kiessling, K. Alt, E. M. Antao, C. Laturnus, I. Diehl, S. Glodde, T. Homeier, U. Bohnke, H. Steinruck, H. C. Philipp, and L. H. Wieler. 2007. Avian pathogenic, uropathogenic, and newborn meningitis-causing *Escherichia coli*: how closely related are they? Int. J. Med. Microbiol. 297:163–176.

23. Garcia-Quintanilla, M., F. Ramos-Morales, and J. Casadesus. 2008. Congugal transfer of the *Salmonella enterica* virulence plasmid in the mouse intestine. J. Bacteriol. 190:1922–1927.

24. Ginns, C. A., M. L. Benham, L. M. Adams, K. G. Whithear, K. A. Bettelheim, B. S. Crabb, and G. F. Browning. 2000. Colonization of the respiratory tract by a virulent strain of avian *Escherichia coli* requires carriage of a conjugative plasmid. Infect. Immun. 68:1535–1541.

25. Gordon, D. M., and C. L. O'Brien. 2006. Bacteriocin diversity and the frequency of multiple bacteriocin production in *Escherichia coli*. Microbiology 152:3239–3244.

26. Griffiths, E. 1999. Iron in biological systems, p. 1–26. In J. Bullen and E. Griffiths (ed.), Iron and infection. Wiley, Chichester, United Kingdom.

27. Hill, V. T., S. M. Townsend, R. S. Arias, J. M. Jenabi, I. Gomez-Gonzalez, H. Shimada, and J. L. Badger. 2004. TraJ-dependent *Escherichia coli* K1 interactions with professional phagocytes are important for early systemic dissemination of infection in the neonatal rat. Infect. Immun. 72:478–488.

28. Holt, D. E., S. Halket, J. de Louvois, and D. Harvey. 2001. Neonatal meningitis in England and Wales: 10 years on. Arch. Dis. Child Fetal Neonatal ed. 84:F85–F89.

29. Houdouin, V., S. Bonacorsi, P. Bidet, J. Blanco, F. De La Rocque, R. Cohen, Y. Aujard, and E. Bingen. 2008. Association between mortality of *Escherichia coli* meningitis in young infants and non-virulent clonal groups of strains. Clin. Microbiol. Infect. 14:685–690.

30. Houdouin, V., S. Bonacorsi, N. Brahimi, O. Clermont, X. Nassif, and E. Bingen. 2002. A uropathogenicity island contributes to the pathogenicity of *Escherichia coli* strains that cause neonatal meningitis. Infect. Immun. 70:5865–5869.

31. Janakiraman, A., and J. M. Slauch. 2000. The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of *Salmonella typhimurium*. Mol. Microbiol. 35:1146–1155.

32. Jeziorski, A., and D. M. Gordon. 2007. Evolution of microcin V and colicin Ia plasmids in *Escherichia coli*. J. Bacteriol. 189:7045–7052.

33. Johnson, J. R., M. A. Kuskowski, T. O'Bryan, and J. N. Maslow. 2002. Epidemiological correlates of virulence genotype and phylogenetic background among *Escherichia coli* blood isolates from adults with diverse-source bacteremia. J. Infect. Dis. 185:1439–1447.

34. Johnson, T. J., S. J. Johnson, and L. K. Nolan. 2006. Complete DNA sequence of a ColBM plasmid from avian pathogenic *Escherichia coli* suggests that it evolved from closely related ColV virulence plasmids. J. Bacteriol. 188:5975–5983.

35. Johnson, T. J., K. E. Siek, S. J. Johnson, and L. K. Nolan. 2006. DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian *Escherichia coli* strains. J. Bacteriol. 188:745–758.

36. Johnson, T. J., Y. Wannemuehler, S. J. Johnson, A. L. Stell, C. Doetkott, J. R. Johnson, K. S. Kim, L. Spanjaard, and L. K. Nolan. 2008. Comparison of extraintestinal pathogenic *Escherichia coli* strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl. Environ. Microbiol. 74:7043–7050.

37. Johnson, T. J., Y. M. Wannemuehler, and L. K. Nolan. 2008. Evolution of the *iss* gene in *Escherichia coli*. Appl. Environ. Microbiol. 74:2360–2369.

38. Kim, K. S. 2003. Pathogenesis of bacterial meningitis: from bacteraemia to neuronal injury. Nat. Rev. Neurosci. 4:376–385.

39. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 5:150–163.

40. Le Gall, T., O. Clermont, S. Gouriou, B. Picard, X. Nassif, E. Denamur, and O. Tenaillon. 2007. Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group *Escherichia coli* strains. Mol. Biol. Evol. 24:2373–2384.

41. McCabe, W. R., B. Kaijser, S. Olling, M. Uwaydah, and L. A. Hanson. 1978. *Escherichia coli* in bacteremia: K and O antigens and serum sensitivity of strains from adults and neonates. J. Infect. Dis. 138:33–41.
other determinants. The pS88 plasmid carries two genes, *iss* and *traT*, which play a role in serum survival and resistance to macrophage phagocytosis, respectively. Several other pS88 coding sequences have no homologs and could also encode new virulence determinants.

In conclusion, pS88 shares a similar structure with avian virulence plasmids, pointing to a possible common ancestor. It is an essential meningeal virulence determinant comparable to the K1 capsule and O45<sub>S88</sub> somatic antigens and is not restricted to the French O45:K1:H7 clone. A comprehensive study of the molecular mechanisms responsible for its virulence may have important implications, possibly beyond the pathogenesis of neonatal meningitis.

REFERENCES

1. Abbott, J. C., D. M. Aanensen, K. Rutherford, S. Butcher, and B. G. Spratt. 2005. WebACT—an online companion for the Artemis Comparison Tool. Bioinformatics 21:3665–3666.

2. Achtman, M., A. Mercer, B. Kusecek, A. Pohl, M. Heuzenroeder, W. Aaronson, A. Sutton, and R. P. Silver. 1983. Six widespread bacterial clones among *Escherichia coli* K1 isolates. Infect. Immun. 39:315–335.

3. Aguero, M. E., and F. C. Cabello. 1983. Relative contribution of ColV plasmid and K1 antigen to the pathogenicity of *Escherichia coli*. Infect. Immun. 40:359–368.

4. Aguero, M. E., G. de la Fuente, E. Vivaldi, and F. Cabello. 1989. ColV increases the virulence of *Escherichia coli* K1 strains in animal models of neonatal meningitis and urinary infection. Med. Microbiol. Immunol. (Berlin) 178:211–216.

5. Badger, J. L., C. A. Wass, S. J. Weissman, and K. S. Kim. 2000. Application of signature-tagged mutagenesis for identification of *Escherichia coli* K1 genes that contribute to invasion of human brain microvascular endothelial cells. Infect. Immun. 68:5056–5061.

6. Bagdasarian, M., A. Bailone, M. M. Bagdasarian, P. A. Manning, R. Lurz, K. N. Timmis, and R. Devoret. 1986. An inhibitor of SOS induction, specified by a plasmid locus in *Escherichia coli*. Proc. Natl. Acad. Sci. USA 83:5723–5726.

7. Barbe, V., D. Vallenet, N. Fonknechten, A. Kreimeyer, S. Oztas, L. Labarre, S. Cruveiller, C. Robert, S. Duprat, P. Wincker, L. N. Ornston, J. Weissenbach, P. Marliere, G. N. Cohen, and C. Medigue. 2004. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 32:5766–5779.

8. Bedford, H., J. de Louvois, S. Halket, C. Peckham, R. Hurley, and D. Harvey. 2001. Meningitis in infancy in England and Wales: follow up at age 5 years. BMJ 323:533–536.

9. Bidet, P., F. Mahjoub-Messai, J. Blanco, J. Blanco, M. Dehem, Y. Aujard, E. Bingen, and S. Bonacorsi. 2007. Combined multilocus sequence typing and O serogrouping distinguishes *Escherichia coli* subtypes associated with infant urosepsis and/or meningitis. J. Infect. Dis. 196:297–303.

10. Bidet, P., A. Metais, F. Mahjoub-Messai, L. Durand, M. Dehem, Y. Aujard, E. Bingen, X. Nassif, and S. Bonacorsi. 2007. Detection and identification by PCR of a highly virulent phylogenetic subgroup among extraintestinal pathogenic *Escherichia coli* B2 strains. Appl. Environ. Microbiol. 73:2373–2377.

11. 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. 177:642–650.

12. Bonacorsi, S., and E. Bingen. 2005. Molecular epidemiology of *Escherichia coli* causing neonatal meningitis. Int. J. Med. Microbiol. 295:373–381.

13. Bonacorsi, S., O. Clermont, V. Houdouin, C. Cordevant, N. Brahimi, A. Marecat, C. Tinsley, X. Nassif, M. Lange, and E. Bingen. 2003. Molecular analysis and experimental virulence of French and North American *Escherichia coli* neonatal meningitis isolates; identification of new virulent clone. J. Infect. Dis. 187:1895–1906.

14. Bonacorsi, S., S. Lefevre, O. Clermont, V. Houdouin, A. Bourrillon, C. Loirat, Y. Aujard, and E. Bingen. 2005. *Escherichia coli* strains causing urinary tract infection in uncircumcised infants resemble urosepsis-like adult strains. J. Urol. 173:195–197.

15. Boyd, E. F., C. W. Hill, S. M. Rich, and D. L. Hartl. 1996. Mosaic structure of plasmids from natural populations of *Escherichia coli*. Genetics 143:1091–1100.

16. Brzuszkiewicz, E., H. Bruggemann, H. Liesegang, M. Emmerth, T. Olschlager, G. Nagy, K. Albermann, C. Wagner, C. Buchrieser, L. Emody, G. Gottschalk, J. Hacker, and U. Dobrindt. 2006. How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic *Escherichia coli* strains. Proc. Natl. Acad. Sci. USA 103:12879–12884.

17. Chuba, P. J., M. A. Leon, A. Banerjee, and S. Palchaudhuri. 1989. Cloning

and DNA sequence of plasmid determinant *iss*, coding for increased serum survival and surface exclusion, which has homology with lambda DNA. Mol. Gen. Genet. 216:287–292.

18. Clermont, O., S. Bonacorsi, and E. Bingen. 2001. The *Yersinia* high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of *Escherichia coli*. FEMS Microbiol. Lett. 196:153–157.

19. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in *Escherichia coli* K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640–6645.

20. de Louvois, J., S. Halket, and D. Harvey. 2005. Neonatal meningitis in England and Wales: sequelae at 5 years of age. Eur. J. Pediatr. 164:730–734.

21. Dozois, C. M., F. Daigle, and R. Curtiss, 3rd. 2003. Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic *Escherichia coli* strain. Proc. Natl. Acad. Sci. USA 100:247–252.

22. Ewers, C., G. Li, H. Wilking, S. Kiessling, K. Alt, E. M. Antao, C. Laturnus, I. Diehl, S. Glodde, T. Homeier, U. Bohnke, H. Steinruck, H. C. Philipp, and L. H. Wieler. 2007. Avian pathogenic, uropathogenic, and newborn meningitis-causing *Escherichia coli*: how closely related are they? Int. J. Med. Microbiol. 297:163–176.

23. Garcia-Quintanilla, M., F. Ramos-Morales, and J. Casadesus. 2008. Congugal transfer of the *Salmonella enterica* virulence plasmid in the mouse intestine. J. Bacteriol. 190:1922–1927.

24. Ginns, C. A., M. L. Benham, L. M. Adams, K. G. Whithear, K. A. Bettelheim, B. S. Crabb, and G. F. Browning. 2000. Colonization of the respiratory tract by a virulent strain of avian *Escherichia coli* requires carriage of a conjugative plasmid. Infect. Immun. 68:1535–1541.

25. Gordon, D. M., and C. L. O'Brien. 2006. Bacteriocin diversity and the frequency of multiple bacteriocin production in *Escherichia coli*. Microbiology 152:3239–3244.

26. Griffiths, E. 1999. Iron in biological systems, p. 1–26. In J. Bullen and E. Griffiths (ed.), Iron and infection. Wiley, Chichester, United Kingdom.

27. Hill, V. T., S. M. Townsend, R. S. Arias, J. M. Jenabi, I. Gomez-Gonzalez, H. Shimada, and J. L. Badger. 2004. TraJ-dependent *Escherichia coli* K1 interactions with professional phagocytes are important for early systemic dissemination of infection in the neonatal rat. Infect. Immun. 72:478–488.

28. Holt, D. E., S. Halket, J. de Louvois, and D. Harvey. 2001. Neonatal meningitis in England and Wales: 10 years on. Arch. Dis. Child Fetal Neonatal ed. 84:F85–F89.

29. Houdouin, V., S. Bonacorsi, P. Bidet, J. Blanco, F. De La Rocque, R. Cohen, Y. Aujard, and E. Bingen. 2008. Association between mortality of *Escherichia coli* meningitis in young infants and non-virulent clonal groups of strains. Clin. Microbiol. Infect. 14:685–690.

30. Houdouin, V., S. Bonacorsi, N. Brahimi, O. Clermont, X. Nassif, and E. Bingen. 2002. A uropathogenicity island contributes to the pathogenicity of *Escherichia coli* strains that cause neonatal meningitis. Infect. Immun. 70:5865–5869.

31. Janakiraman, A., and J. M. Slauch. 2000. The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of *Salmonella typhimurium*. Mol. Microbiol. 35:1146–1155.

32. Jeziorowski, A., and D. M. Gordon. 2007. Evolution of microcin V and colicin Ia plasmids in *Escherichia coli*. J. Bacteriol. 189:7045–7052.

33. Johnson, J. R., M. A. Kuskowski, T. O'Bryan, and J. N. Maslow. 2002. Epidemiological correlates of virulence genotype and phylogenetic background among *Escherichia coli* blood isolates from adults with diverse-source bacteremia. J. Infect. Dis. 185:1439–1447.

34. Johnson, T. J., S. J. Johnson, and L. K. Nolan. 2006. Complete DNA sequence of a ColBM plasmid from avian pathogenic *Escherichia coli* suggests that it evolved from closely related ColV virulence plasmids. J. Bacteriol. 188:5975–5983.

35. Johnson, T. J., K. E. Siek, S. J. Johnson, and L. K. Nolan. 2006. DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian *Escherichia coli* strains. J. Bacteriol. 188:745–758.

36. Johnson, T. J., Y. Wannemuehler, S. J. Johnson, A. L. Stell, C. Doetkott, J. R. Johnson, K. S. Kim, L. Spanjaard, and L. K. Nolan. 2008. Comparison of extraintestinal pathogenic *Escherichia coli* strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl. Environ. Microbiol. 74:7043–7050.

37. Johnson, T. J., Y. M. Wannemuehler, and L. K. Nolan. 2008. Evolution of the *iss* gene in *Escherichia coli*. Appl. Environ. Microbiol. 74:2360–2369.

38. Kim, K. S. 2003. Pathogenesis of bacterial meningitis: from bacteraemia to neuronal injury. Nat. Rev. Neurosci. 4:376–385.

39. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 5:150–163.

40. Le Gall, T., O. Clermont, S. Gouriou, B. Picard, X. Nassif, E. Denamur, and O. Tenaillon. 2007. Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group *Escherichia coli* strains. Mol. Biol. Evol. 24:2373–2384.

41. McCabe, W. R., B. Kaijser, S. Olling, M. Uwaydah, and L. A. Hanson. 1978. *Escherichia coli* in bacteremia: K and O antigens and serum sensitivity of strains from adults and neonates. J. Infect. Dis. 138:33–41.
42. Mercer, A. A., G. Morelli, M. Heuzenroeder, M. Kamke, and M. Achtman. 1984. Conservation of plasmids among *Escherichia coli* K1 isolates of diverse origins. Infect. Immun. **46**:649–657.

43. Montgomerie, J. Z., A. Bindereif, J. B. Neilands, G. M. Kalmanson, and L. B. Guze. 1984. Association of hydroxamate siderophore (aerobactin) with *Escherichia coli* isolated from patients with bacteremia. Infect. Immun. **46**:835–838.

44. Morales, C., M. D. Lee, C. Hofacre, and J. J. Maurer. 2004. Detection of a novel virulence gene and a *Salmonella* virulence homologue among *Escherichia coli* isolated from broiler chickens. Foodborne Pathog. Dis. **1**:160–165.

45. Mori, H., A. Kondo, A. Ohshima, T. Ogura, and S. Hiraga. 1986. Structure and function of the F plasmid genes essential for partitioning. J. Mol. Biol. **192**:1–15.

46. Moulin-Schouleur, M., C. Schouler, P. Tailliez, M. R. Kao, A. Bree, P. Germon, E. Oswald, J. Mainil, M. Blanco, and J. Blanco. 2006. Common virulence factors and genetic relationships between O18:K1:H7 *Escherichia coli* isolates of human and avian origin. J. Clin. Microbiol. **44**:3484–3492.

47. Negre, V. L., S. Bonacorsi, S. Schubert, P. Bidet, X. Nassif, and E. Bingen. 2004. The siderophore receptor IroN, but not the high-pathogenicity island or the hemin receptor ChuA, contributes to the bacteremic step of *Escherichia coli* neonatal meningitis. Infect. Immun. **72**:1216–1220.

48. Ott, M., L. Bender, G. Blum, M. Schmittroth, M. Achtman, H. Tschape, and J. Hacker. 1991. Virulence patterns and long-range genetic mapping of extraintestinal *Escherichia coli* K1, K5, and K100 isolates: use of pulsed-field gel electrophoresis. Infect. Immun. **59**:2664–2672.

49. Picard, B., C. Journet-Mancy, N. Picard-Pasquier, and P. Goulet. 1993. Genetic structures of the B2 and B1 *Escherichia coli* strains responsible for extra-intestinal infections. J. Gen. Microbiol. **139**:3079–3088.

50. Plainvert, C., P. Bidet, C. Peigne, V. Barbe, C. Medigue, E. Denamur, E. Bingen, and S. Bonacorsi. 2007. A new O-antigen gene cluster has a key role in the virulence of the *Escherichia coli* meningitis clone O45:K1:H7. J. Bacteriol. **189**:8528–8536.

51. Ruppe, E., P. Bidet, C. Verdet, G. Arlet, and E. Bingen. 2006. First detection of the Ambler class C 1 AmpC beta-lactamase in *Citrobacter freundii* by a new, simple double-disk synergy test. J. Clin. Microbiol. **44**:4204–4207.

52. Sabri, M., M. Caza, J. Proulx, M. H. Lymberopoulos, A. Bree, M. Moulin-Schouleur, R. Curtis III, and C. M. Dozois. 2008. Contribution of the SitABCD, MntH, and FeoB metal transporters to the virulence of avian pathogenic *Escherichia coli* O78 strain chi7122. Infect. Immun. **76**:601–611.

53. Sabri, M., S. Leveille, and C. M. Dozois. 2006. A SitABCD homologue from an avian pathogenic *Escherichia coli* strain mediates transport of iron and manganese and resistance to hydrogen peroxide. Microbiology **152**:745–758.

54. Sansonetti, P. J., D. J. Kopecko, and S. B. Formal. 1981. *Shigella sonnei* plasmids: evidence that a large plasmid is necessary for virulence. Infect. Immun. **34**:75–83.

55. Saul, D., A. J. Spiers, J. McAnulty, M. G. Gibbs, P. L. Bergquist, and D. F. Hill. 1989. Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids. J. Bacteriol. **171**:2697–2707.

56. Skyberg, J. A., T. J. Johnson, J. R. Johnson, C. Clabots, C. M. Logue, and L. K. Nolan. 2006. Acquisition of avian pathogenic *Escherichia coli* plasmids by a commensal *E. coli* isolate enhances its abilities to kill chicken embryos, grow in human urine, and colonize the murine kidney. Infect. Immun. **74**: 6287–6292.

57. Smith, H. W., and M. B. Huggins. 1976. Further observations on the association of the colicine V plasmid of *Escherichia coli* with pathogenicity and with survival in the alimentary tract. J. Gen. Microbiol. **92**:335–350.

58. Stumpe, S., R. Schmid, D. L. Stephens, G. Georgiou, and E. P. Bakker. 1998. Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of *Escherichia coli*. J. Bacteriol. **180**:4002–4006.

59. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. **22**:4673–4680.

60. Tivendale, K. A., J. L. Allen, C. A. Ginns, B. S. Crabb, and G. F. Browning. 2004. Association of *iss* and *iucA*, but not *tsh*, with plasmid-mediated virulence of avian pathogenic *Escherichia coli*. Infect. Immun. **72**:6554–6560.

61. Tschäpe, H., H. Steinrück, P. Buchholz, R. Prager, E. Tietze, G. Seltmann, and J. Hacker. 1990. Molecular analysis of *Escherichia coli* from neonatal infections and its epidemiological implication, p. 224–234. *In* E. Gravel, L. Stern, I. Syllim-Rapoport, and R. Waver (ed.), Research in perinatal medicine II. Verlag Gesundheit, Berlin, Germany.

62. Unhanand, M., M. M. Mustafa, G. H. McCracken, Jr., and J. D. Nelson. 1993. Gram-negative enteric bacillary meningitis: a twenty-one-year experience. J. Pediatr. **122**:15–21.

63. Vallenet, D., L. Labarre, Z. Rouy, V. Barbe, S. Bocs, S. Cruveiller, A. Lajus, G. Pascal, C. Scarpelli, and C. Medigue. 2006. MaGe: a microbial genome annotation system supported by synteny results. Nucleic Acids Res. **34**:53–65.

64. Waters, V. L., and J. H. Crosa. 1991. Colicin V virulence plasmids. Microbiol. Rev. **55**:437–450.

65. Welch, R. A., V. Burland, G. Plunkett, 3rd, P. Redford, P. Roesch, D. Rasko, E. L. Buckles, S. R. Liou, A. Boutin, J. Hackett, D. Stroud, G. F. Mayhew, D. J. Rose, S. Zhou, D. C. Schwartz, N. T. Perna, H. L. Mobley, M. S. Donnenberg, and F. R. Blattner. 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic *Escherichia coli*. Proc. Natl. Acad. Sci. USA **99**:17020–17024.

66. Williams, P. H. 1979. Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of *Escherichia coli*. Infect. Immun. **26**:925–932.

67. Wullenweber, M., L. Beutin, S. Zimmermann, and C. Jonas. 1993. Influence of some bacterial and host factors on colonization and invasiveness of *Escherichia coli* K1 in neonatal rats. Infect. Immun. **61**:2138–2144.
42. Mercer, A. A., G. Morelli, M. Heuzenroeder, M. Kamke, and M. Achtman. 1984. Conservation of plasmids among *Escherichia coli* K1 isolates of diverse origins. Infect. Immun. **46**:649–657.

43. Montgomerie, J. Z., A. Bindereif, J. B. Neilands, G. M. Kalmanson, and L. B. Guze. 1984. Association of hydroxamate siderophore (aerobactin) with *Escherichia coli* isolated from patients with bacteremia. Infect. Immun. **46**:835–838.

44. Morales, C., M. D. Lee, C. Hofacre, and J. J. Maurer. 2004. Detection of a novel virulence gene and a *Salmonella* virulence homologue among *Escherichia coli* isolated from broiler chickens. Foodborne Pathog. Dis. **1**:160–165.

45. Mori, H., A. Kondo, A. Ohshima, T. Ogura, and S. Hiraga. 1986. Structure and function of the F plasmid genes essential for partitioning. J. Mol. Biol. **192**:1–15.

46. Moulin-Schouleur, M., C. Schouler, P. Tailliez, M. R. Kao, A. Bree, P. Germon, E. Oswald, J. Mainil, M. Blanco, and J. Blanco. 2006. Common virulence factors and genetic relationships between O18:K1:H7 *Escherichia coli* isolates of human and avian origin. J. Clin. Microbiol. **44**:3484–3492.

47. Negre, V. L., S. Bonacorsi, S. Schubert, P. Bidet, X. Nassif, and E. Bingen. 2004. The siderophore receptor IroN, but not the high-pathogenicity island or the hemin receptor ChuA, contributes to the bacteremic step of *Escherichia coli* neonatal meningitis. Infect. Immun. **72**:1216–1220.

48. Ott, M., L. Bender, G. Blum, M. Schmittroth, M. Achtman, H. Tschape, and J. Hacker. 1991. Virulence patterns and long-range genetic mapping of extraintestinal *Escherichia coli* K1, K5, and K100 isolates: use of pulsed-field gel electrophoresis. Infect. Immun. **59**:2664–2672.

49. Picard, B., C. Journet-Mancy, N. Picard-Pasquier, and P. Goulet. 1993. Genetic structures of the B2 and B1 *Escherichia coli* strains responsible for extra-intestinal infections. J. Gen. Microbiol. **139**:3079–3088.

50. Plainvert, C., P. Bidet, C. Peigne, V. Barbe, C. Medigue, E. Denamur, E. Bingen, and S. Bonacorsi. 2007. A new O-antigen gene cluster has a key role in the virulence of the *Escherichia coli* meningitis clone O45:K1:H7. J. Bacteriol. **189**:8528–8536.

51. Ruppe, E., P. Bidet, C. Verdet, G. Arlet, and E. Bingen. 2006. First detection of the Ambler class C 1 AmpC beta-lactamase in *Citrobacter freundii* by a new, simple double-disk synergy test. J. Clin. Microbiol. **44**:4204–4207.

52. Sabri, M., M. Caza, J. Proulx, M. H. Lymberopoulos, A. Bree, M. Moulin-Schouleur, R. Curtis III, and C. M. Dozois. 2008. Contribution of the SitABCD, MntH, and FeoB metal transporters to the virulence of avian pathogenic *Escherichia coli* O78 strain chi7122. Infect. Immun. **76**:601–611.

53. Sabri, M., S. Leveille, and C. M. Dozois. 2006. A SitABCD homologue from an avian pathogenic *Escherichia coli* strain mediates transport of iron and manganese and resistance to hydrogen peroxide. Microbiology **152**:745–758.

54. Sansonetti, P. J., D. J. Kopecko, and S. B. Formal. 1981. *Shigella sonnei* plasmids: evidence that a large plasmid is necessary for virulence. Infect. Immun. **34**:75–83.

55. Saul, D., A. J. Spiers, J. McAnulty, M. G. Gibbs, P. L. Bergquist, and D. F. Hill. 1989. Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids. J. Bacteriol. **171**:2697–2707.

56. Skyberg, J. A., T. J. Johnson, J. R. Johnson, C. Clabots, C. M. Logue, and L. K. Nolan. 2006. Acquisition of avian pathogenic *Escherichia coli* plasmids by a commensal *E. coli* isolate enhances its abilities to kill chicken embryos, grow in human urine, and colonize the murine kidney. Infect. Immun. **74**: 6287–6292.

57. Smith, H. W., and M. B. Huggins. 1976. Further observations on the association of the colicine V plasmid of *Escherichia coli* with pathogenicity and with survival in the alimentary tract. J. Gen. Microbiol. **92**:335–350.

58. Stumpe, S., R. Schmid, D. L. Stephens, G. Georgiou, and E. P. Bakker. 1998. Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of *Escherichia coli*. J. Bacteriol. **180**:4002–4006.

59. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. **22**:4673–4680.

60. Tivendale, K. A., J. L. Allen, C. A. Ginns, B. S. Crabb, and G. F. Browning. 2004. Association of *iss* and *iucA*, but not *tsh*, with plasmid-mediated virulence of avian pathogenic *Escherichia coli*. Infect. Immun. **72**:6554–6560.

61. Tschäpe, H., H. Steinrück, P. Buchholz, R. Prager, E. Tietze, G. Seltmann, and J. Hacker. 1990. Molecular analysis of *Escherichia coli* from neonatal infections and its epidemiological implication, p. 224–234. *In* E. Gravel, L. Stern, I. Syllim-Rapoport, and R. Waver (ed.), Research in perinatal medicine II. Verlag Gesundheit, Berlin, Germany.

62. Unhanand, M., M. M. Mustafa, G. H. McCracken, Jr., and J. D. Nelson. 1993. Gram-negative enteric bacillary meningitis: a twenty-one-year experience. J. Pediatr. **122**:15–21.

63. Vallenet, D., L. Labarre, Z. Rouy, V. Barbe, S. Bocs, S. Cruveiller, A. Lajus, G. Pascal, C. Scarpelli, and C. Medigue. 2006. MaGe: a microbial genome annotation system supported by synteny results. Nucleic Acids Res. **34**:53–65.

64. Waters, V. L., and J. H. Crosa. 1991. Colicin V virulence plasmids. Microbiol. Rev. **55**:437–450.

65. Welch, R. A., V. Burland, G. Plunkett, 3rd, P. Redford, P. Roesch, D. Rasko, E. L. Buckles, S. R. Liou, A. Boutin, J. Hackett, D. Stroud, G. F. Mayhew, D. J. Rose, S. Zhou, D. C. Schwartz, N. T. Perna, H. L. Mobley, M. S. Donnenberg, and F. R. Blattner. 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic *Escherichia coli*. Proc. Natl. Acad. Sci. USA **99**:17020–17024.

66. Williams, P. H. 1979. Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of *Escherichia coli*. Infect. Immun. **26**:925–932.

67. Wullenweber, M., L. Beutin, S. Zimmermann, and C. Jonas. 1993. Influence of some bacterial and host factors on colonization and invasiveness of *Escherichia coli* K1 in neonatal rats. Infect. Immun. **61**:2138–2144.

Editor: F. C. Fang