Adhesion and Invasion Characteristics of a Septicaemic Avian Escherichia coli Strain Are Plasmid Mediated

Eliana Guedes Stehling, PhD

Tatiana Amabile de Campos, MSc

Alessandra Ferreira, BSc

Wanderley Dias da Silveira, PhD

Department of Microbiology and Immunology, Biology Institute, Campinas State University,

Campinas, Brazil.

This study was supported by grants no 96/03683-0 and 99/05830-2 from the Foundation for the Support of Research of the State of São Paulo (FAPESP) and no 300121/90-3 from the National Council for Scientific and Technological Development (CNPq).

KEY WORDS: Escherichia coli, adhesion, fimbriae, plasmids

Abstract

A strain of Escherichia coli isolated from chicken with septicemia, denominated SEPT13, was studied and analyzed according to its pathogenic characteristics. This strain presents a lethal dose (DL) 50% of 4.0 x 105 CFU/mL, expresses Ia, Ib, E1, E3, K, and B colicins and aerobactin, is resistant to ampicillin and streptomycin, possesses DNA sequences related to fimA, csgA, and tsh genes, is able to adhere and to invade Hep-2 and trachea cells, expresses fimbriae as observed by electron microscopy, and have 5 plasmids with 2.7, 4.7, 43, 56, and 88 MDa. The 43 MDa plasmid was transferred to a nonpathogenic receptor strain using a conjugation assay, originating the transconjugant E, that maintained the adhesion and invasion capacity. This transconjugant was mutagenized with transposon TnphoA, generating the mutant Mut05, which lost its capacity to adhere and invade Hep-2 cells and to adhere to trachea cells. These results lead us to propose that 43-MDa plasmid could be responsible for the adhesion and invasion capacities present in the wild type strain (SEPT 13).

INTRODUCTION

Escherichia coli is believed to be a member of the normal bowel flora of mammals and birds, but some strains are pathogenic because of the acquisition of virulence factors.1 E. coli infection is usually seen as a potentially fatal septicemia secondary to a virus respiratory disease,2 and its presence greatly increases the severity of the disease.3

Avian pathogenic E. coli (APEC) strains belong mainly to serogroups O1, O2, and O78 and have the capacity to express several potential virulence factors.4 One of these factors is the expression of adhesins, which are fundamental for the adherence capacity of these strains to the epithelium of the respiratory tract of chickens.2,3,5 Several types of adhesins have been shown to exist in APEC strains (type 1 and type P fimbriae and tsh adhesin). Type 1 and P fimbriae are the ones that could be directly related to the pathogenic process. Type 1 fimbriae, which is encoded by the fim gene cluster, is located at 98 minutes on the E. coli chromosome, 6 being expressed mainly in the trachea, lung, and air sac.7 However, the role of type P fimbriae in the pathogenicity of avian E. coli has not yet been fully elucided.8 The tsh gene, as suggested by Provence and Curtiss,9 could act as an adhesin in the initial stages of colonization of the avian respiratory tract because of its hemagglutinating capacity. Its possible relation with the APEC strains pathogenic process was suggested by Maurer et al.,10 who detected its presence in approximately 46% of clinical avian E. coli isolates but not among commensal isolates. Dozois et al.11 showed that the tsh gene was located in a ColV plasmid. Other virulence factors such as colicins and aerobactin are also described as having a role during the pathogenic process of APEC.12,13

The purpose of this work was to study the expression of several biological characteristics, such as the adhesion and invasion capacities of cells cultivated in vitro, adhesion to trachea epithelial cells, colicin and aerobactin production, and fimbriae expression by an avian septicaemic E. coli strain (SEPT13). Additionally, we sought to correlate these characteristics with the presence of plasmids that are harbored by this strain.

MATERIAL AND METHODS

Bacterial strains and growth media

Strain SEPT13 is an avian pathogenic E. coli (APEC) strain isolated from a chicken showing clinical signs of septicemia. E. coli strains MS101 (nonpathogenic, nalidixic acid resistant) and HB101 (nonfimbriated, streptomycin resistant) were used as recipient strains for conjugation and transformation experiments. E. coli strain LG 1522,14 was used as an indicator strain for aerobactin production. E. coli strains R80 (all colicins ), R81 (col I), R82 (col Ia), R83 (col Ib), R675 (col E1), R676 (col E3), R914 (col ROW-K), R915 (col V), and R996 (col B) were used as indicator strains for specific colicins and were a gift from Dr. E. C. Souza from the University of Minas Gerais at Belo Horizonte. Plasmid pRA1 of 86 MDa and plasmids of 32, 5.12, 3.48, 3.03, 2.24, 1.69, 1.51, and 1.25 MDa harbored by strain V517 were a gift from Dr. James B. Kaper (CVD-USA). These plasmids were used as molecular standards in agarose gel electrophoresis. Plasmid pRT733,15 was used for mutagenesis technique. Media LB and LA were used for bacterial growth. All strains were stored in LB medium containing 15% glycerol at -70˚C to avoid plasmid losses.

Pathogenicity Assay

Pathogenicity assays were performed as described by Fantinatti et al.16 Briefly, a 1.0-mL suspension (LB medium, 37˚C, 14 to 18 hours; washed twice with and resuspended into 0.85% sterilized saline solution) of the strain to be tested was diluted tenfold (10-1 to 10-11) and 0.5 mL of each dilution were subcutaneously injected into the neck region of groups of 6 1-day-old male chickens. These groups were observed throughout a 7-day period, and the LD50% was calculated by the method of Reed and Muench17 for each strain.

Determination of Antibiotics
Resistance Levels

The resistance of antimicrobial drugs (ampicillin [Amp], kanamycin [Km], streptomycin [Sm], tetracycline [Tc], and chloramphenicol [Cm]) was determined as described by Chulasari and Suthienkul.18 Concentrations of 5, 10, 25, 50, 100, 250, and 500 mg/mL were used to determine the resistance level for each antibiotic. The maximum concentration that still presented bacterial growth was considered to be the minimal inhibitory concentration for that antibiotic.

Colicin Production

Colicin production was determined as described by Fantinatti et al.16 Briefly, strains were cultivated overnight in LB medium at 37˚C and plated onto LA agar. After overnight incubation at 37˚C, the bacterial growth was killed under chloroform fumes and overlaid with 3.0 mL of soft LA medium containing a colicin-indicator strain. The capacity for colicin production was determined by the presence of a clear halo around the killed bacterial colony after an overnight incubation period.

Aerobactin Production

Aerobactin production was assayed by the method of Carbonetti and Williams,14 using E. coli LG 1522 as the indicator strain. For this purpose, symmetric holes were made to LA medium containing 200 mmol/L of a-a-dipiridyl and filled with the supernatant of the bacterial growth (iron-free LB medium, 37˚C, overnight) of each strain to be tested. Once the medium had absorbed all the liquid, strain LG 1522 was inoculated onto the surface of it, and the petri dish was incubated at 37˚C overnight. Growth of LG 1522 colonies around a given hole indicated the capacity of that strain to produce aerobactin.

Adhesion and Invasion Capacities of Hep-2 Cells and Adhesion to Trachea Epithelial Cells

Adhesion and invasion capacity assays were performed as described by Scaletsky et al.19 and Vidotto et al.1 respectively.

The capacity to adhere to trachea epithelial cells was tested as described by Dho and Lafont5 and Pourbakhsh et al.17 For this purpose, 19-day-old avian SPF embryoned eggs, supplied by the Fort Dodge Company, were used.

Plasmid DNA extraction and agarose
gel electrophoresis

Plasmid DNA was extracted as described by Sambrook et al,20 resuspended into sterilized deionized water, and stored frozen until use. Plasmid DNA to be used in the transformation experiments was cleaned using the Wizard DNA Clean-up columns (Promega). Plasmid DNA electrophoresis and ethidium bromide staining of the gels were accomplished as described by Sambrook et al.20

Conjugation Experiments

Conjugation assays were performed as described by Azevedo and da Costa.21 For this purpose, a donor strain grown until log phase (LB medium, 37˚C, with no agitation) was mixed with equal amount of recipient strain grown until the plato phase in the same conditions over 0.02 mm millipore membrane onto LA medium. The mixture was kept at 37˚C overnight, resuspended into LB medium, diluted (1:10 dilutions) into 0.9% sterilized saline, and plated onto LA medium containing antibiotics for selecting recombinant strains.

Transformation Experiments and Transposon Mutagenesis

Transformation assays were performed as described by Sambrook et al.20 Transposon mutageneis (TnphoA) was performed as described by Taylor et al.15 using plasmid pRT733. Mutations were obtained using LA medium containing 40 mg/mL of 5-bromo-4 chloro-3-indolyl phosphate and selective antibiotics. Blue kanamycin colonies were analyzed using electrophoresis of plasmid DNA profiles.

Detection of fimA, csgA, papA, and tsh Sequences by PCR

Two mL of genomic bacterial DNA, extracted as described by Ausubel et al22 and resuspended in TE buffer and 10 mg/mL of RNAse, were used for PCR reactions. Primers for fimA, csgA, papA, and tsh sequences were those described by Marc et al,23 Maurer et al,10 Vidotto et al,8 and Maurer et al,10 respectively. The PCR reaction conditions were the same as described by these authors.

Electron Microscopy Studies

For electron microscopy studies, a donor strain was grown in LB medium at 37˚C overnight. After centrifugation (13,000 g for 30 seconds), the sediment was resuspended in 200 mL of milli-Q water, and 10 mL of this growth were mixed and fixed with 1% phosphotungstic acid (PTA) for 30 seconds. This technique was performed in a 400-mesh grid coated with Formvar. After 24 hours, the grids were dried in a carbon-evaporator and observed in a transmission electron microscope (LEO 906).

Tnphoa Molecular Probe and Hybridization With Plasmid DNA

A 3450-bp fragment of transposon TnphoA was cut from plasmid pRT733 using restriction enzyme BstE II and purified from agarose gel using the dialysis method described by Sambrook et al.20 This fragment was labeled using alkaline phosphatase and hybridized with plasmid DNA using the Amersham Pharmacia Alk Phos system.

RESULTS

The biological characteristics of strain SEPT13 and its derivative recombinant strains are shown in Table 1. Strain SEPT13 is an APEC strain that was isolated from the liver of a chicken presenting clinical signs of septicemia. It is resistant to streptomicin (Sm) and ampicillin (Ap), produces colicins Ia, Ib, E1, E3, K, and B and the siderophore aerobactin. This strain presents D-mannose-resistant diffuse adhesion (DA) and invasion of Hep-2 cells cultivated in vitro (Figure 1) and adhesion on trachea epithelial cells (Figure 2), and harbored 5 different plasmids of 2.7, 4.7, 43, 56, and 88 MDa (Figure 3). The PCR reaction demonstrated the presence of fimA, csgA and tsh genes in this strain (results not shown).

Transconjugant strain TE was obtained after conjugation of strain SEPT13 with strain MS101. It is resistant to Ap and NA, does not produces colicins or aerobactin; presents D-mannose-resistant diffuse adhesion on Hep-2 cells cultivated in vitro and adhesion on trachea epithelial cells. It is also invasive to Hep-2 cells (Figure 1). Agarose gel electrophoresis demonstrated that plasmid of 43 MDa was transferred to the recipient strain MS101 (Figure 3). The PCR reaction demonstrated the presence of tsh gene in this transconjugant (Figure 4).

Strain Mut05 was obtained by transposon mutagenesis (TnphoA) of strain TE. This strain is resistant to Ap, NA and kanamicin (Km) and lost adhesion and invasion capacities on Hep-2 cells as well as the adhesion capacity on trachea epithelial cells that were present in strain E. Agarose gel electrophoresis showed that transposon TnphoA was inserted into the 43-MDa plasmid (Figure 3). Hybridization experiments using a 3450-bp BstII fragment of transposon TnphoA as a molecular probe confirmed the insertion of this transposon in the 43-MDa plasmid (results not shown). This mutation also amplified the tsh gene in the PCR reaction (Figure 4).

Electron microscopy was performed on strains SEPT13, TE, Mut05, and MS101. Because the strains MS101 and Mut05 expressed fimbriae, the plasmids of 43-MDa and 48-MDa from strains TE and Mut05, respectively, were transferred by transformation experiments to a nonfimbriated, nonpathogenic, Sm-resistant strain (HB101). This was performed to verify if the fimbriae initially present in strains TE and Mut05 was either the same one expressed by strain SEPT13 or was another one that was expressed by strain MS101. The obtained recombinant strain harboring the plasmid 43-MDa had the same adhesive and invasive characteristics as observed for strain TE, and the recombinant strain harboring the 48-MDa plasmid did not show these traits. The electron microscope studies showed that the recombinant strain with the 43-MDa plasmid was expressing fimbriae, and the one with the 48-MDa plasmid was not (Figure 5).

DISCUSSION

To correlate the presence of plasmids with virulence and pathogenicity of avian septicemic strain SEPT13, we studied all the possible traits that could be related to the virulence of this strain. Through conjugation, one 43-MDa plasmid was transferred to a nonpathogenic receptor strain and the obtained transconjugant did not produce either colicin or aerobactin, but was able to adhere to trachea cells and adhere and invade Hep-2 cells. However, it was not pathogenic in the 1-day-old chicken assay. After transposon mutagenesis, this recombinant strain lost the ability to adhere and invade the cell types discussed.

The wild type strain (SEPT13) did not express colicin V. The expression of colicin V is correlated with the presence of a plasmid that would carry pathogenic traits.24 The SEPT13 strain, although pathogenic, is not a colicin V producer. However, it produces other colicin types. This could not be ascribed to the 43-MDa plasmid, because transconjugant TE did not produce any colicin type. Similar results were also seen with aerobactin production.

The adhesion capacity to Hep-2 and trachea cells, observed in the transconjugant E, could be related to the 43-MDa plasmid. In studies with E. coli in human infections, some plasmids were also believed to be responsible for the adhesion capacity. Baldini et al25 described a plasmid with 50 to 70 MDa, encountered in enteropathogenic E. coli. This plasmid was believed to be responsible for the capacity of this strain to adhere in Hep-2 cells.

Donnemberg et al26 described a 60-MDa plasmid denominated EAF, which is responsible for the capacity of one strain of E. coli (EPEC) to adhere to in vitro cultivated cells in a localized manner. This plasmid has the bfpa genes that codifies the type IV fimbriae.

Adhesins with a potential role in the pathogenic mechanisms of APEC strains include F1, P, and Curli fimbriae and also the temperature-sensitive hemagglutinin (Tsh).10 Among these adhesins, Tsh was described by Provence and Curtiss19 as an outer membrane protein with the ability to agglutinate chicken erythrocytes and show homology to the serine-type immunoglobulin A1 (IgA1) proteases produced by Haemophilus influenzae and Neisseria gonorrhoeae. Dozois et al12 suggested that this protein could be directly or indirectly responsible for adherence to the host erythrocytes and could also act as an important adhesin for the initial stages of colonization of the avian respiratory tract.

In this study, the mutation 05 lost the ability to adhere and invade in vitro cultivated cells and adhere to trachea epithelial cells. However, it had the tsh gene amplified by the PCR technique, as did the wild type strain (SEPT13) and the transconjugant strain TE. These results indicate that either the Tsh adhesin is not related to the adhesion capacity of transconjugant TE, since the mut05 does not have a transposon insertion in the tsh gene, or it is responsible for these characteristics but the transposon needed another essential gene for expression of Tsh adhesin.

Another factor that could be responsible for the adhesion and invasion capacities could be the small and thin fimbriae expressed by the transconjugant TE. This fimbriae does not resemble type 1, P, or Curli fimbriae, which are described as being the main adhesins of APEC strains. This conclusion is based on the fact that PCR reactions using the transconjugant TE did not amplify the fimA (type 1 fimbriae), papA (type P fimbriae), and csgA (Curli fimbriae) genes. Although not identified, the fimbriae expressed by strain TE could be responsible for the adhesion mechanisms encountered in strain SEPT13 and transconjugant TE. This hypothesis is strengthened by the facts that mutant 05 lost the capacity to express any recognizable fimbriae, as seen on electron microscopy, and that it had lost its adhesion and invasion capacities.

Also, although 43 MDa plasmid has genes related to the adhesion and invasion capacities, it does not have genes responsible for direct pathogenic capacity. This is noted because the obtained transconjugants were not pathogenic in the 1-day-old chickens assay (LD50% > 1011CFU/mL). These results suggest that the entire pathogenic process present in the SEPT13 strain is probably dependent on the coordinate expression of multiple genes. These genes could be localized either in different plasmids or in plasmids and in the chromosome.

To better understand this process, we are studying the other plasmids harbored by the strain SEPT13 and sequencing the plasmid of 43 MDa.

ACKNOWLEDGMENTS

This work was supported by grants No 98/03683-0 and No 99/05830-2 from The Foundation for the Support of Research of the State of São Paulo (FAPESP) and No 300121/90-3 from The National Council for Scientific and Technological Development (CNPq).

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Table 1. Biological Characteristics of Strains SEPT13 and its Derivatives

PCR

LD 50% Adhesion Adhesion Antibiotic amplified Plasmids

Strains CFU/mL (Hep-2 cells) (trachea cells) Colicins Resistance genes (MDa)

SEPT13 4.0 ¥ 105 DA + Ia, Ib, Ap, Sm fimA, 43, 56,

E1, E3, csgA, tsh 88

K, B

TE >1011 DA + - Ap, NA tsh 43

Mut05 >1011 - - - Ap, NA, Km tsh 48

MS101 >1011 - - - NA - -

DA, diffuse adhesion; Ap, ampicillin; Sm, streptomicin; NA, nalidixic acid; Km, kanamicin.

Figure 1. Adhesion and invasion capacities of strain SEPT13 and its derivative recombinant strains on Hep-2 cells. (A) adhesion in D-mannose absence, strain SEPT13. (B) Adhesion in D-mannose presence, strain SEPT13. (C) Invasion in D-mannose absence, strain SEPT13. (D) Invasion in D-mannose presence, strain SEPT13. (E) Adhesion in D-mannose presence, strain TE. (F) Invasion in D-mannose presence, strain TE. (G) Mut05. (H) MS101. Magnification: x 1000.

Figure 2. Adhesion of the SEPT13 and its derivative recombinant strains in trachea cells. (A) Strain SEPT13. (B) Strain MS101. (C) Strain TE. (D) Mut05. Magnification: x 1000.

Figure 3. Agarose gel (0.7%) electrophoresis of plasmid DNA of the SEPT13 strain and its derivative recombinant strains and reference plasmids. (A) Plasmid V517 (32 MDa). (B) Plasmid pRA1 (86 MDa). (C) Strain SEPT13. (D) Strain TE. (E) Mut05.

Figure 4. Agarose gel (1.3%) electrophoresis of the PCR reaction for amplification of the tsh gene in the SEPT13 strain and its derivative recombinant strains. (A) Molecular weight markers (1 Kb). (B) Strain SEPT13. (C) Strain E. (D) Mut05. (E) Strain MS101.

Figure 5. Electron micrograph of the SEPT13 and its derivatives recombinant strains. (A) Strain SEPT13. (B) Strain HB101. (C) Recombinant TE. (D) Recombinant Mut05.