Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnson, T. J. Articles by Nolan, L. K. Search for Related Content PubMed PubMed Citation Articles by Johnson, T. J. Articles by Nolan, L. K.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, November 2005, p. 4681-4688, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4681-4688.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid

Timothy J. Johnson, Kylie E. Siek, Sara J. Johnson, and Lisa K. Nolan^*

Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, 1802 Elwood Drive, VMRI #2, Ames, Iowa 50011

Received 9 May 2005/ Returned for modification 13 July 2005/ Accepted 9 August 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, a 101-kb IncF plasmid from an avian pathogenic Escherichia coli (APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the E. coli F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal E. coli strain from an apparently healthy bird, a Salmonella enterica serovar Typhimurium strain, and a uropathogenic E. coli strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antimicrobial resistance among bacterial pathogens of food animals can complicate veterinary therapy. Resistant animal pathogens may also be a threat to human health if these resistant bacteria enter the food supply or otherwise serve as reservoirs of resistance genes for human pathogens. Transmissible R plasmids that encode multidrug resistance would seem a likely means by which animal pathogens could acquire resistance genes or transmit them to human pathogens. This study examines an R plasmid encoding multidrug resistance in an avian pathogenic Escherichia coli (APEC) isolate. APEC strains are important and prevalent bacterial pathogens of poultry (3) and are frequently found to be resistant to multiple antimicrobial agents (21, 37), including ampicillin, tetracycline, aminoglycosides, fluoroquinolones, quaternary ammonium compounds, and heavy metals (37). Genes encoding such resistance are often found on large, transmissible R plasmids (20). Not surprisingly, multidrug-resistant APEC strains often carry conjugative plasmids (8). Interestingly, plasmids have been shown to be transferable from poultry to human isolates (23), suggesting that APEC strains and their plasmids might serve as reservoirs of resistance genes for bacteria that affect public health. In the present study, the first complete sequence of a transmissible APEC R plasmid is presented and analyzed. Additionally, an effort was made to determine the transmissibility of this plasmid to other bacteria found in poultry and to an E. coli strain from human disease in order to assess the potential of this plasmid to serve as a reservoir of resistance genes for pathogens of animal and human health significance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids. The original source of pAPEC-O2-R, the plasmid sequenced in this study, was a wild-type avian E. coli isolate named APEC O2, with the "O2" in its name referring to its serogroup. APEC O2 was isolated from a chicken clinically diagnosed with colibacillosis. All strains were grown at 37°C in Luria-Bertani broth medium (LB broth; Difco Laboratories, Detroit, MI), supplemented as needed with antimicrobial agents at the following concentrations: ampicillin, 100 µg/ml; tetracycline, 12.5 µg/ml; and/or nalidixic acid, 30 µg/ml. All bacterial strains were stored at –70°C in brain heart infusion broth (Difco Laboratories) with 10% glycerol until they were used (32). The recipients used in the conjugation studies included avian pathogenic E. coli strain 419; an avian fecal commensal E. coli (AFEC) isolate from an apparently healthy chicken, A3; a uropathogenic E. coli (UPEC) strain, 2000-1; and Salmonella enteric serovar Typhimurium strain 475. Additional details about these recipients are provided in Table 1.

View this table: [in this window] [in a new window]

TABLE 1. Bacterial strains used in matings with APEC O2

Antimicrobial susceptibility testing. The donor strain possessing pAPEC-O2-R, the recipient strains, and their transconjugants were examined for resistance to ampicillin, tetracycline, chloramphenicol, streptomycin, spectinomycin, sulfisoxazole, gentamicin, trimethoprim, silver nitrate, and benzalkonium chloride by disk diffusion assays. These assays were performed with BBL Sensi-Disk antimicrobial susceptibility test disks (BD, Franklin Lakes, NJ), in accordance with the CLSI (formerly the NCCLS) standard Kirby-Bauer disk diffusion method (28, 29). Briefly, Mueller-Hinton agar plates (Difco Laboratories) were swabbed with E. coli cultures grown to a McFarland standard of 0.5. Zones of inhibition were measured in millimeters (including disk diameter) and were categorized as sensitive or resistant according to the CLSI breakpoints.

Disk diffusion was also used to test the E. coli isolates for their susceptibilities to benzalkonium chloride and silver nitrate. For these compounds, sterile 5.5-cm filter paper disks (Fisher Scientific) were placed on Mueller-Hinton agar plates swabbed with E. coli cultures grown to a McFarland standard of 0.5. Ten microliters of either of these compounds was then pipetted onto an individual disk from the following stock concentrations: 0.1 M silver nitrate and 0.1 M benzalkonium chloride. All plates were incubated overnight at 37°C, and zones of inhibition were measured in millimeters and compared to known positive and negative controls on the following day. The positive control used to measure susceptibility to benzalkonium chloride and silver nitrate was APEC O2, which is resistant to these agents. E. coli DH5, which is sensitive to these two antimicrobial agents, was used as a negative control (31). Strains were classified as sensitive or resistant to benzalkonium chloride and silver nitrate based on comparison to those of known positive and negative controls.

Bacterial conjugations and DNA isolation. The transmissibility of pAPEC-O2-R was determined by mating APEC O2 with several plasmid-less bacteria (Table 1) by using a previously described protocol (19). Mating mixtures were incubated overnight at 25°C, 37°C, and 42°C; and transconjugants were selected on Mueller-Hinton agar (Difco Laboratories) containing appropriate antibiotics. Putative transconjugants were verified by their antimicrobial resistance profiles, plasmid contents, and gene contents, as determined by the use of a series of multiplex PCR protocols described previously (30). Mating frequencies were determined by measuring the proportion of transconjugant colonies to recipient colonies. The plasmid DNA used in this study was obtained from overnight cultures in LB broth containing ampicillin (100 µg/ml), according to the methods of Wang and Rossman (36). Plasmid DNA was separated by horizontal agarose gel electrophoresis (0.7% TAE [Tris-acetate-EDTA]; 3.5 V/cm).

Shotgun library construction and sequencing. Plasmid DNA was sheared, concentrated, and desalted by using standard protocols (31). DNA was end repaired (30 min; 15°C; 100-µl reaction mixture consisting of 2 µg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 µM each deoxynucleoside triphosphate, 10 µl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min; 50°C; 100-µl reaction mixture consisting of 2 µg sheared DNA; 50 µM each dCTP, dGTP, and dTTP; 2 mM dATP; 20 U Taq polymerase [MBI Fermentas], 10 µl Yellow Tango buffer). The A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified by standard methods (31) prior to cloning into pGEM-T (Promega, Madison, WI).

Sequencing was performed by MWG Biotech, Inc. (Hedersberg, Germany). Briefly, plasmid clones were grown for 20 h in 1.8 ml LB broth supplemented with 200 µg/ml ampicillin in deep-well boxes. Plasmid DNA were prepared on a RoboPrep2500 DNA-Prep-Robot (MWG-Biotech, Ebersberg, Germany) by using a NucleoSpin Robot-96 Plasmid kit (Macherey & Nagel, Dueren, Germany) and sequenced from both ends with standard primers by using the BigDye Terminator chemistry (Applied Biosystems, Foster City, CA). The data were collected with ABI 3700 and ABI 3730xl capillary sequencers (Applied Biosystems) and assembled by using the Gap 4 program (5).

Analysis and annotation. Open reading frames (ORFs) in the plasmid sequence were identified by using GeneQuest from DNASTAR (Madison, WI) and GLIMMER 2.02 (11), followed by manual inspection. Translated ORFs were then compared to known protein sequences by using the BLAST program (March 2005 version; National Center for Biotechnology Information). Those with greater than 60% identity were considered matches. Hypothetical proteins with greater than 60% identity to one or more previously published proteins were classified as conserved hypothetical proteins, and ORFs with less than 60% identity to any published sequences were classified as hypothetical proteins. The G+C contents of individual ORFs were analyzed by using GeneQuest (DNASTAR). Insertion sequences and repetitive elements were identified by using IS FINDER (http://www-is.biotoul.fr/). Genomic comparisons of pAPEC-O2-R to similar plasmids were done by using MAUVE alignments (10). Amino acid sequence alignments were performed by using MegAlign (DNASTAR).

Nucleotide sequence accession number. The complete sequence of pAPEC-O2-R was deposited in GenBank under accession number AY214164.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antimicrobial susceptibility testing. The transconjugant containing pAPEC-O2-R and plasmid donor APEC O2 were resistant to ampicillin, sulfisoxazole, tetracycline, streptomycin, gentamicin, trimethoprim, silver nitrate, and benzalkonium chloride; the recipient, E. coli DH5, was susceptible to all antimicrobial agents tested. APEC O2 was mated to several plasmid-less strains of enteric bacteria, including AFEC A3, APEC 419, S. enterica serovar Typhimurium 475, and UPEC 2000-1. All pairings produced transconjugants at similar mating frequencies (Table 1). In each case, the recipients acquired the resistance profiles of the donor (Table 1) and a large plasmid consistent with the size of pAPEC-O2-R.

Sequencing and analysis of pAPEC-O2-R. Three thousand ninety-five shotgun clones of pAPEC-O2-R were arrayed, sequenced, and assembled by using the Gap4 program (5). The assembly resulted in the generation of a complete circular sequence (Fig. 1) of 101,375 bp with approximately 20-fold coverage. pAPEC-O2-R contains 123 predicted ORFs; all coding regions and their closest database matches are provided in Table 2. One hundred eleven of these ORFs showed 60% or greater identity to a previously published sequence. Of these, 82 have a known function, and 29 are conserved hypothetical proteins. The remaining 12 ORFs are classified as hypothetical proteins for which no significant matches in the database were identified. Overall, these ORFs were arranged in distinct regions and encoded antimicrobial resistance, transmissibility, replication, and maintenance (Fig. 1).

View larger version (34K): [in this window] [in a new window]

FIG. 1. Circular genetic map of pAPEC-O2-R. Coding regions are indicated by arrows pointing in the direction of transcription. Yellow arrows indicate coding regions involved in antimicrobial resistance, blue arrows indicate coding regions involved in replication, red and pink arrows indicate coding regions involved in plasmid transfer, brown arrows indicate coding regions involved in plasmid maintenance, green arrows indicate mobile elements, blue-gray arrows indicate conserved hypothetical proteins, and gray arrows indicate unknown hypothetical proteins.

View this table: [in this window] [in a new window]

TABLE 2. Coding regions of pAPEC-O2-R

Analysis of the coding regions of pAPEC-O2-R revealed a 33,950-bp region containing 15 genes responsible for resistance to at least eight antimicrobial agents (Table 2). This region begins following the hnh gene with the start of the sil gene cluster, a seven-component system that encodes resistance to silver and other heavy metals (16). Following this cluster is an insertion sequence, IS26, that marks the beginning of the tetAR complex encoding tetracycline resistance. Immediately following the tetAR genes is a 12,282-bp region of pAPEC-O2-R that contains a class 1 integron also found in transposon Tn21 (24). The class 1 integron of pAPEC-O2-R contains three gene cassettes, including the catB3, aadA5, and folA genes. Following the class 1 integron is Tn3, a transposon containing bla_TEM-1, a gene encoding a beta-lactamase.

pAPEC-O2-R also contains genes involved in its own maintenance and replication. Near the transfer region are several genes involved in plasmid maintenance, including hok and sok, ssb, psiA, stbA, stbB, parA, and psiB (13). Four replication genes, repA1 to repA4, are also found on pAPEC-O2-R.

The average G+C content of pAPEC-O2-R is 53%, which is similar to that of the E. coli K-12 genome (4). However, several regions have notable deviations from this G+C ratio (Fig. 2). The transfer region has an average G+C content of 52%, which is markedly different from those of its flanking plasmid maintenance and gene cassette-containing regions, with G+C contents of 56% and 57%, respectively. These two regions are separated by the silver resistance operon, which has an average G+C content of 51%.

View larger version (42K): [in this window] [in a new window]

FIG. 2. Analysis of G+C contents of coding regions of pAPEC-O2-R. The dashed line represents the average G+C content of the E. coli K-12 genome (4).

Comparative genomics. pAPEC-O2-R was compared to similar IncF plasmids whose complete sequences are available. pAPEC-O2-R was compared to E. coli plasmids R100 (GenBank accession no. NC_002134) and C15-1a (6), its two closest DNA sequence matches in the National Center for Biotechnology Information database. Comparison of translated coding sequences revealed that 27% of the 201 total predicted proteins were common to all three plasmids, 19% were shared by two of the three plasmids, and 54% were present in only one of the three plasmids. Most of the proteins common to the three plasmids were components of the transfer and plasmid maintenance regions of pAPEC-O2-R. By using a MAUVE alignment (10), the complete sequence of pAPEC-O2-R was aligned with the sequences of E. coli plasmids F (14), R100 (accession no. GenBank NC_002134), 1658/97 (accession no. GenBank NC_004998), and C15-1a (6). The alignments of these five plasmids identified a common backbone containing genes involved in plasmid transfer, maintenance, and replication. The proteins within this backbone account for approximately 40% of the total proteins within pAPEC-O2-R. The remainder of these plasmids appear to be composed primarily of antimicrobial resistance genes, mobile elements, and hypothetical proteins of unknown function.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Large plasmids are common among APEC strains and contain genes important to antimicrobial resistance (8) and virulence (12, 17, 19, 30). In this study, the first complete sequence of an APEC plasmid is presented. pAPEC-O2-R was found to contain a functional multidrug resistance-determining region, as acquisition of pAPEC-O2-R by the recipients was accompanied by acquisition of the donor strain's antimicrobial resistance pattern. This resistance region contains the sil gene cluster, which encodes resistance to silver and other heavy metals and which has previously been identified on large plasmids in Salmonella (16), Serattia (15), and Klebsiella spp. (9). Also, within this region of pAPEC-O2-R are what appear to be remnants of Tn21, a transposon coined the "flagship of the floating genome" for its ability to facilitate the acquisition and/or the deletion of resistance genes within the bacterial genome (24). Tn21 has previously been identified in APEC (24). The Tn21-like region of pAPEC-O2-R contains an intact class 1 integron previously ascribed to Tn21, named In2, and the 5' portions of Tn21. However, unlike the previously described structure of Tn21 (24), the class 1 integron in pAPEC-O2-R lacks the operon encoding mercury resistance on its 3' end. Nevertheless, the presence of a class 1 integron and other components of Tn21 within this region of pAPEC-O2-R indicates that portions of this region might be derived from Tn21. The class 1 integron of pAPEC-O2-R contains three gene cassettes, including catB3 (7), which encodes resistance to chloramphenicol; aadA5 (33), which contributes to aminoglycoside resistance; and folA (1, 2), which encodes resistance to trimethoprim. All resistance genes on pAPEC-O2-R appear to be functional, as determined by disk diffusion, with the exception of the catB3 gene encoding chloramphenicol resistance. Only an intermediate zone of inhibition was obtained when strains containing pAPEC-O2-R were grown in the presence of chloramphenicol disks. Analysis of the gene cassette region of the class 1 integron on pAPEC-O2-R identified a 132-bp attC site on the 3' end of folA, a 60-bp attC site on the 3' end of catB3, and a 57-bp attC site on the 3' end of aadA5. No promoter sequences were identified for any individual gene cassettes; only a common promoter within the intI1 gene was identified. This class 1 integron is also flanked on its 3' conserved end by an intact Tn3, which contains bla_TEM-1, and on its 5' end are other remnants of Tn21, which is downstream of the silver resistance-determining operon.

Overall, the arrangement of the antimicrobial resistance region of pAPEC-O2-R is unique compared to that in other R plasmids. Several plasmids that encode resistance to multiple heavy metals and toxins have been sequenced, such as plasmid R478 in Serratia marcescens (15) and plasmid LVPK in Klebsiella pneumoniae (9), but they lack the class 1 integron of pAPEC-O2-R. Alternatively, several E. coli R plasmids that contain Tn21-like regions have been sequenced, such as plasmids R100 (GenBank accession no. NC_002134), C15-1a (6), and 1658/97 (GenBank accession no. NC_004998); but these plasmids lack the heavy metal resistance genes found in pAPEC-O2-R. Therefore, the composition of pAPEC-O2-R is noteworthy due to its diversity and its large number of resistance genes.

In addition to its functional multidrug resistance-encoding region, pAPEC-O2-R possesses a 31,887-bp transfer region nearly identical to that found in several E. coli plasmids, including the F plasmid (14) and R100 (GenBank accession no. NC_002134). This region is also similar to the transfer region of a large plasmid (pSLT) found in an S. enterica serovar Typhimurium strain (27). This transfer region encodes a type 4 secretion system that facilitates conjugative transfer (22). The transfer region of pAPEC-O2-R is functional, as evidenced by the fact that pAPEC-O2-R is transmissible by conjugation into commensal and pathogenic bacteria, such as E. coli and S. enterica serovar Typhimurium, that may be found in the poultry production environment. Therefore, it is possible that plasmid transfer might occur naturally in the poultry environment. Indeed, studies have shown that large plasmids are common among avian E. coli strains (12, 30) and that these plasmid-containing E. coli strains may be transmitted between birds (23). Interestingly, such transfer may also occur from birds to humans (23). In the present study, transfer of pAPEC-O2-R from APEC O2 to a human UPEC strain occurred in vitro, supporting the possibility that R plasmids harbored by animal pathogens may be reservoirs of resistance genes for human pathogens.

pAPEC-O2-R also contains genes involved in its own maintenance. Flanking the transfer region are two genes, hok and sok (for host killing and suppression of killing, respectively), involved in postsegregational killing of plasmid-free cells, thus ensuring that pAPEC-O2-R is retained during cell replication (13). Also within this region are ssb, psiA, and psiB, which may be involved in the conjugal transfer of pAPEC-O2-R into a recipient cell, with psiB inhibiting the cellular SOS response upon transfer, thus protecting the single-stranded plasmid DNA in the recipient prior to the synthesis of the second strand (25). Three more genes, stbA, stbB, and parA, also lie within this plasmid maintenance region and are involved in partitioning of pAPEC-O2-R into daughter cells during cell division, thus playing a role in plasmid stability (35). The presence of an active partitioning system and an antisense RNA-regulated plasmid addiction system on pAPEC-O2-R ensures that this plasmid is retained by bacterial populations, even in the absence of selective pressures within the poultry environment. Thus, these plasmids may have emerged in populations of APEC due to some type of selective pressure, such as the use of antimicrobials in the poultry environment, and they are likely retained by these APEC strains, even in the absence of this selective pressure, due to their active partitioning and plasmid addiction systems.

Additionally, pAPEC-O2-R contains four coding regions, repA1 to repA4, that are likely involved in replication, copy number, and stability. BLAST analysis of these coding regions shows that they are very similar to those of IncF plasmids, a diverse group of plasmids with similar replicons and transfer regions (Table 2). The replicons included in this group are RepFIIA, whose members include pR100 and pR1; RepFIC, which is a replicon of the F plasmid; RepFIB, a replicon of ColV plasmids such as pRK100 (34); and RepFIII, a close relative of RepFII that includes E. coli plasmid SU316 (26). Comparison of the four predicted replication proteins in pAPEC-O2-R with those of pR100 (GenBank accession no. NC_002134), pRK100 (34), and pSU316 (26) revealed that pAPEC-O2-R shares the strongest identity with pR100, an IncFII plasmid. The repA1-coding sequence, which is directly involved in plasmid replication, and repA4, a gene immediately adjacent to the origin of replication that is involved in plasmid stability (18), appear to be highly conserved (99% protein identity). The repA2- and repA3-coding sequences, which are involved in replication control, were quite different among the four plasmids analyzed, exhibiting only partial protein identity to published sequences (Table 2). Others have also reported that these portions of IncF replicons are areas of nonhomology (26). However, these coding regions in pAPEC-O2-R are considerably different from any sequences published to date. Further work is required to determine the significance of these differences.

In summary, a 101-kb IncF plasmid from an APEC strain was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, contains genes for plasmid maintenance and replication. It also has a functional transfer region that allows its transmission to bacterial strains that are found in the poultry environment or that cause human infection. Additionally, pAPEC-O2-R contains an antimicrobial resistance-encoding region that encodes multidrug resistance. This region of the plasmid is unique among previously described IncF plasmids, as it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. Differences in the G+C contents of individual ORFs suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health importance.

 
This project was funded in part by the Roy J. Carver Charitable Trust Fund.

 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, 1802 Elwood Drive, VMRI #2, Ames, IA 50011. Phone: (515) 294-3534. Fax: (515) 294-3839. E-mail: lknolan{at}iastate.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Adrian, P. V., C. J. Thomson, K. P. Klugman, and S. G. Amyes. 2000. New gene cassettes for trimethoprim resistance, dfr13, and streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron. Antimicrob. Agents Chemother. 44:355-361.[Abstract/Free Full Text]
2
2. Barclay, B. J., T. Huang, M. G. Nagel, V. L. Misener, J. C. Game, and G. M. Wahl. 1988. Mapping and sequencing of the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae. Gene 63:175-185.[CrossRef][Medline]
3
3. Barnes, H. J., and W. B. Gross. 1997. Colibacillosis, p. 131-141. In B. W. Calnek (ed.), Diseases of poultry, 10th ed. Iowa State University Press, Ames.
4
4. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-1474.[Abstract/Free Full Text]
5
5. Bonfield, J. K., K. Smith, and R. Staden. 1995. A new DNA assembly program. Nucleic Acids Res. 22:4992-4999.
6
6. Boyd, D. A., S. Tyler, S. Christianson, A. McGeer, M. P. Muller, B. M. Willey, E. Bryce, M. Gardam, P. Nordmann, and M. R. Mulvey. 2004. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob. Agents Chemother. 48:3758-3764.[Abstract/Free Full Text]
7
7. Bunny, K. L., R. M. Hall, and H. W. Stokes. 1995. New mobile gene cassettes containing an aminoglycoside resistance gene, aacA7, and a chloramphenicol resistance gene, catB3, in an integron in pBWH301. Antimicrob. Agents Chemother. 3:686-693.
8
8. Caudry, S. D., and V. A. Stanisich. 1979. Incidence of antibiotic-resistant Escherichia coli associated with frozen chicken carcasses and characterization of conjugative R plasmids derived from such strains. Antimicrob. Agents Chemother. 16:701-709.[Abstract/Free Full Text]
9
9. Chen, Y. T., H. Y. Chang, Y. C. Lai, C. C. Pan, S. F. Tsai, and H. L. Peng. 2004. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. Gene 337:189-198.[CrossRef][Medline]
10
10. Darling, A. C., B. Mau, F. R. Blattner, and N. T. Perna. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14:1394-1403.[Abstract/Free Full Text]
11
11. Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636-4641.[Abstract/Free Full Text]
12
12. Doetkott, D. M., L. K. Nolan, C. W. Giddings, and D. L. Berryhill. 1996. Large plasmids of avian Escherichia coli isolates. Avian Dis. 40:927-930.[CrossRef][Medline]
13
13. Franch, T., A. P. Gultyaev, and K. Gerdes. 1997. Programmed cell death by hok/sok of plasmid R1: Processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding. J. Mol. Biol. 273:38-51.[CrossRef][Medline]
14
14. Frost, L. S., K. Ippen-Ihler, and R. A. Skurray. 1994. Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol. Rev. 58:162-210.[Abstract/Free Full Text]
15
15. Gilmour, M. W., N. R. Thomson, M. Sanders, J. Parkhill, and D. E. Taylor. 2004. The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics. Plasmid 52:182-202.[CrossRef][Medline]
16
16. Gupta, A., K. Matsui, J. F. Lo, and S. Silver. 1999. Molecular basis for resistance to silver cations in Salmonella. Nat. Med. 5:183-188.[CrossRef][Medline]
17
17. Ike, K., K. Kawahara, H. Danbara, and K. Hume. 1992. Serum resistance and aerobactin iron uptake in avian Escherichia coli mediated by conjugative 100-megadalton plasmid. J. Vet. Med. Sci. 54:1091-1098.[Medline]
18
18. Jiang, T., Y. N. Min, W. Liu, D. D. Womble, and R. H. Rownd. 1993. Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance. J. Bacteriol. 175:5350-5358.[Abstract/Free Full Text]
19
19. Johnson, T. J., C. W. Giddings, S. M. Horne, P. S. Gibbs, R. E. Wooley, J. Skyberg, P. Olah, R. Kercher, J. S. Sherwood, S. L. Foley, and L. K. Nolan. 2002. Location of increased serum survival gene and selected virulence traits on a conjugative R plasmid in an avian Escherichia coli isolate. Avian Dis. 46:342-352.[CrossRef][Medline]
20
20. Koh, C. L., and C. H. Kok. 1984. Antimicrobial resistance and conjugative R plasmids in Escherichia coli strains isolated from animals in peninsular Malaysia. Southeast Asian Trop. Med. Public Health 1:37-43.
21
21. Lanz, R., P. Kuhnert, and P. Boerlin. 2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet. Microbiol. 91:73-84.[CrossRef][Medline]
22
22. Lawley, T. D., W. A. Klimke, M. J. Gubbins, and L. S. Frost. 2003. F factor conjugation is a true type IV secretion system. FEMS Microbiol. Lett. 224:1-15.[CrossRef][Medline]
23
23. Levy, S. B., G. B. Fitzgerald, and A. B. Macone. 1976. Spread of antibiotic-resistant plasmids from chicken to chicken and from chicken to man. Nature 260:40-42.[CrossRef][Medline]
24
24. Liebert, C. A., R. M. Hall, and A. O. Summers. 1999. Transposon Tn21, flagship of the floating genome. Microbiol. Mol. Biol. Rev. 63:507-522.[Abstract/Free Full Text]
25
25. Loh, S., R. Skurray, J. Celerier, M. Bagdasarian, A. Bailone, and R. Devoret. 1990. Nucleotide sequence of the psiA (plasmid SOS inhibition) gene located on the leading region of plasmids F and R6-5. Nucleic Acids Res. 18:4597.[Free Full Text]
26
26. Lopez, J., P. Crespo, J. C. Rodriquez, I. Andres, and J. M. Ortiz. 1989. Analysis of IncF plasmids evolution: nucleotide sequence of an IncFIII replication region. Gene 78:183-187.[CrossRef][Medline]
27
27. McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, Du, F., S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-856.[CrossRef][Medline]
28
28. National Committee for Clinical Laboratory Standards. 1997. Approved standard M2-A6. Performance standards for antimicrobial disk susceptibility tests, 6th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
29
29. National Committee for Clinical Laboratory Standards. 1999. NCCLS document M100-S9. Performance standards for antimicrobial susceptibility testing, 9th ed. Informational supplement. National Committee for Clinical Laboratory Standards, Wayne, Pa.
30
30. Rodriguez-Siek, K. E., C. W. Giddings, C. Doetkott, T. J. Johnson, and L. K. Nolan. 2005. Characterizing the APEC pathotype. Vet. Res. 2:241-256.
31
31. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
32
32. Sanderson, K. E., and D. R. Zeigler. 1991. Storing, shipping, and maintaining records on bacterial strains. Methods Enzymol. 204:248-264.[CrossRef][Medline]
33
33. Sandvang, D. 1999. Novel streptomycin and spectinomycin resistance gene as a gene cassette within a class 1 integron isolated from Escherichia coli. Antimicrob. Agents Chemother. 43:3036-3038.[Abstract/Free Full Text]
34
34. Starcic-Erjavec, M., W. Gaastra, J. van Putten, and D. Zgur-Bertok. 2003. Identification of the origin of replications and partial characterization of plasmid pRK100. Plasmid 50:102-112.[CrossRef][Medline]
35
35. Tabuchi, A., Y. Min, D. D. Womble, and R. H. Rownd. 1992. Autoregulation of the stability operon of IncFII plasmid NR1. J. Bacteriol. 174:7629-7634.[Abstract/Free Full Text]
36
36. Wang, Z., and T. G. Rossman. 1994. Large-scale supercoiled plasmid preparation by acidic phenol extraction. BioTechniques 16:460-463.[Medline]
37
37. Yang, H., S. Chen, D. G. White, S. Zhao, P. McDermott, R. Walker, and J. Meng. 2004. Characterization of multiple-antimicrobial-resistant Escherichia coli isolates from diseased chickens and swine in China. J. Clin. Microbiol. 42:3483-3489.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, November 2005, p. 4681-4688, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4681-4688.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Bonnet, C., Diarrassouba, F., Brousseau, R., Masson, L., Topp, E., Diarra, M. S. (2009). Pathotype and Antibiotic Resistance Gene Distributions of Escherichia coli Isolates from Broiler Chickens Raised on Antimicrobial-Supplemented Diets. Appl. Environ. Microbiol. 75: 6955-6962 [Abstract] [Full Text]  
• Jamalludeen, N., She, Y.-M., Lingohr, E. J., Griffiths, M. (2009). Isolation and characterization of virulent bacteriophages against Escherichia coli serogroups O1, O2, and O78. Poult. Sci. 88: 1694-1702 [Abstract] [Full Text]  
• Lewinson, O., Lee, A. T., Rees, D. C. (2009). A P-type ATPase importer that discriminates between essential and toxic transition metals. Proc. Natl. Acad. Sci. USA 106: 4677-4682 [Abstract] [Full Text]  
• Rasko, D. A., Rosovitz, M. J., Myers, G. S. A., Mongodin, E. F., Fricke, W. F., Gajer, P., Crabtree, J., Sebaihia, M., Thomson, N. R., Chaudhuri, R., Henderson, I. R., Sperandio, V., Ravel, J. (2008). The Pangenome Structure of Escherichia coli: Comparative Genomic Analysis of E. coli Commensal and Pathogenic Isolates. J. Bacteriol. 190: 6881-6893 [Abstract] [Full Text]  
• Fricke, W. F., Wright, M. S., Lindell, A. H., Harkins, D. M., Baker-Austin, C., Ravel, J., Stepanauskas, R. (2008). Insights into the Environmental Resistance Gene Pool from the Genome Sequence of the Multidrug-Resistant Environmental Isolate Escherichia coli SMS-3-5. J. Bacteriol. 190: 6779-6794 [Abstract] [Full Text]  
• Joo, L. M., Macfarlane-Smith, L. R., Okeke, I. N. (2007). Error-Prone DNA Repair System in Enteroaggregative Escherichia coli Identified by Subtractive Hybridization. J. Bacteriol. 189: 3793-3803 [Abstract] [Full Text]  
• Johnson, T. J., Wannemuehler, Y. M., Johnson, S. J., Logue, C. M., White, D. G., Doetkott, C., Nolan, L. K. (2007). Plasmid Replicon Typing of Commensal and Pathogenic Escherichia coli Isolates. Appl. Environ. Microbiol. 73: 1976-1983 [Abstract] [Full Text]  
• Johnson, T. J., Wannemeuhler, Y. M., Scaccianoce, J. A., Johnson, S. J., Nolan, L. K. (2006). Complete DNA Sequence, Comparative Genomics, and Prevalence of an IncHI2 Plasmid Occurring among Extraintestinal Pathogenic Escherichia coli Isolates. Antimicrob. Agents Chemother. 50: 3929-3933 [Abstract] [Full Text]  
• Skyberg, J. A., Johnson, T. J., Johnson, J. R., Clabots, C., Logue, C. M., Nolan, L. K. (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 [Abstract] [Full Text]  
• Sunde, M., Norstrom, M. (2006). The prevalence of, associations between and conjugal transfer of antibiotic resistance genes in Escherichia coli isolated from Norwegian meat and meat products. J Antimicrob Chemother 58: 741-747 [Abstract] [Full Text]  
• Johnson, T. J., Siek, K. E., Johnson, S. J., Nolan, L. K. (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 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnson, T. J. Articles by Nolan, L. K. Search for Related Content PubMed PubMed Citation Articles by Johnson, T. J. Articles by Nolan, L. K.