SEARCH

SEARCH BY CITATION

Keywords:

  • Escherichia hermannii;
  • biofilm;
  • Wzt;
  • apical periodontitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Escherichia hermannii, formerly classified as enteric group 11 of Escherichia coli, is considered to be nonpathogenic. In this report, we described some of the pathogenic properties of a viscous material-producing E. hermannii strain YS-11, which was clinically isolated from a persistent apical periodontitis lesion. YS-11 possessed cell surface-associated meshwork-like structures that are found in some biofilm-forming bacteria and its viscous materials contained mannose-rich exopolysaccharides. To further examine the biological effect of the extracellular viscous materials and the meshwork structures, we constructed a number of mutants using transposon mutagenesis. Strain 455, which has a transposon inserted into wzt, a gene that encodes an ATP-binding cassette transporter, lacked the expression of the cell surface-associated meshwork structures and the ability to produce extracellular materials. Complementation of the disrupted wzt in strain 455 with an intact wzt resulted in the restoration of these phenotypes. We also compared these strains in terms of their ability to induce abscess formation in mice as an indication of their pathogenicity. Strains with meshwork-like structures induced greater abscesses than those induced by strains that lacked such structures. These results suggest that the ability to produce mannose-rich exopolysaccharides and to form meshwork-like structures on E. hermannii might contribute to its pathogenicity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Escherichia hermannii was formerly classified as enteric group 11 of Escherichia coli, and reclassified as a distinct species in 1982 within the Escherichia genus on the basis of DNA–DNA relatedness (Brenner et al., 1982). Escherichia hermannii is distinguished from E. coli by its production of a yellow pigment and by various biochemical characteristics including the fermentation of cellobiose and a positive reaction to KCN (Brenner et al., 1982). Escherichia hermannii is considered to be nonpathogenic, although a few clinical cases of infection are associated with this bacterium, such as infections of human wounds (Pien et al., 1985), a cephalohematoma of a neonate (Dahl et al., 2002), a case of sepsis with duodenal perforation in a neonate (Ginsberg & Daum, 1987), diarrheal stools (Chaudhury et al., 1999), and purulent conjunctivitis (Poulou et al., 2008). A low-level resistance of E. hermannii against amoxicillin and ticarcillin by its production of β-lactamase (HER-1) has also been described (Fitoussi et al., 1995; Beauchef-Havard et al., 2003). Isolation of E. hermannii from contaminated soils at an oil refinery suggests that this organism has an environmental habitat and can survive under adverse environmental conditions (Hernandez et al., 1998). However, the association of this organism with oral infections has not been reported thus far.

Some strains of E. hermannii are also known to yield false-positive results in serological tests directed against E. coli O157:H7, Yersinia enterocolitica serotype O:9, Brucella melitensis, Brucella abortus, Vibrio cholerae O1, and Salmonella group N (O:30). This is because the lipopolysaccharides of these bacteria contain perosamine as a common antigenic O-chain (Perry & Bundle, 1990; Rice et al., 1992; Godfroid et al., 1998; Reeves & Wang, 2002; Munoz et al., 2005).

In this report, we have determined some of the pathogenic properties of a clinical isolate of E. hermannii obtained from an infected root canal of a persistent apical periodontitis lesion (Chavez de Paz, 2007; Yamane et al., 2009). Random insertion mutagenesis using the EZ-Tn5〈KAN-2〉 transposon revealed that a gene cluster encoded in the wzt (a gene for an ATPase domain protein Wzt) of the ATP-binding cassette (ABC)-type transporter (Davidson & Chen, 2004) in the perosamine biosynthesis system could be involved in the biofilm formation by this organism.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Bacteria and culture conditions

A clinical strain capable of producing viscous materials was isolated from a persistent apical periodontitis lesion. The isolate was designated as YS-11 and was the primary strain used in this study. YS-11 was identified in our laboratory as E. hermannii by 16S rRNA gene sequencing. The nucleotide sequence of the 16S rRNA gene [DNA Data Bank of Japan (DDBJ) accession: AB377402; http://www.ddbj.nig.ac.jp] was identical to that of E. hermannii GTC347 (DDBJ accession: AB273738). This was confirmed by PCR amplification of a bla gene encoding E. hermannii class A β-lactamase (HER-1) using the methodology as detailed elsewhere (Beauchef-Havard et al., 2003). The nucleotide sequence obtained from YS-11 (DDBJ accession: AB479111) showed 100% similarity to E. hermannii blaHER-1 (EMBL accession: AF311385). Stock cultures of YS-11 and E. hermannii ATCC33650 (a reference strain for E. hermannii) were grown on trypticase soy agar (BBL Microbiology Systems, Cockeysville, ND) supplemented with 0.5% yeast extract (Difco Laboratories, Detroit, MI) (TSAY) or grown in a trypticase soy broth supplemented with 0.5% yeast extract (TSBY). Bacterial cultures were grown aerobically at 37 °C in an incubation chamber.

Viscosity of spent culture media and biofilm-like structures of YS-11

YS-11 and ATCC33650 were grown in TSBY for 12 h and the viscosity of the spent culture media was measured using a rotary viscometer as described previously (Yamanaka et al., 2009). Briefly, the culture medium (550 μL) was placed into a rotor, and the viscosity was measured as shearing stress between a rotor and a rotor shaft at 50 r.p.m., 20 °C, using a rotary viscometer (Tokimec Inc., Tokyo, Japan). Five independent cultures of each strain were measured and statistical differences between the two groups were determined using an unpaired t-test with the level of significance set at P<0.05.

To examine cell surface structures, scanning electron microscopy (SEM) was performed. Bacteria grown on TSAY for 24 h were collected on a piece of filter paper (Glass fiber GA55, Toyo Roshi, Tochigi, Japan), fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 h and 1% OsO4 in 0.1 M phosphate buffer for 1 h at 4 °C, and dehydrated through an ethanol series and 2-methyl-2-propanol, followed by platinum ion coating (E-1030, Hitachi, Tokyo, Japan). Specimens were examined using an SEM (S-4800, Hitachi) at an accelerating voltage of 3 kV.

Chemical composition of viscous materials

The exopolysaccharide was prepared from culture supernatants as described previously (Yamanaka et al., 2009). In brief, YS-11 was grown at 37 °C in TSBY for 24 h. Supernatants were separated by centrifuging the liquid culture at 12 000 g for 30 min, and sodium acetate was added to a final concentration of 5%. The mixture was stirred for 30 min at 22 °C, and the exopolysaccharide was isolated by ethanol precipitation from the reaction mixture. The ethanol-precipitated material was collected by centrifugation (18 200 g for 15 min at 22 °C), dissolved in 5% sodium acetate, and treated with chloroform : 1-butanol (1 : 5 by volume). Water-soluble and chloroform-butanol layers were separated by centrifugation. An equal amount of ethanol was added to the water-soluble fraction to isolate the exopolysaccharides. This procedure was repeated twice, and the ethanol-precipitated material was freeze-dried and stored at −80 °C until use (Campbell & Pappenheimer, 1966). Contaminated lipopolysaccharides were removed from preparations using the method described by Adam et al. (1995).

The sugar composition of the purified viscous material was determined by means of HPLC for neutral and amino sugars and colorimetry for uronic acid as detailed elsewhere (Yamanaka et al., 2009).

Preparation of competent YS-11 cells and random insertion mutagenesis

To examine the biological effect of extracellular viscous materials and cell surface-associated structures, mutants lacking these phenotypes were constructed by transposon mutagenesis.

Fifteen milliliters of an overnight culture of YS-11 was used to inoculate 800 mL of TSBY. The culture was incubated at 37 °C until the OD600 nm of the bacterial culture measured reached 0.6–0.7. The cells were harvested by centrifugation at 5700 g for 20 min at 4 °C and washed three times with ice-cold 10% glycerol. The cells were resuspended with 4 mL of 10% ice-cold glycerol, divided into small aliquots, frozen with dry ice-100% cold ethanol, and stored at −80 °C until use.

Random insertion mutagenesis was carried out using the EZ-Tn5〈KAN-2〉 Tnp Transposome Kit (EZ-Tn5 Tnp; Epicentre Biotechnologies, Madison, WI). One microliter of EZ-Tn5 Tnp was mixed with 50 μL of the competent cells of YS-11. The mixture was placed in an ice-cold 2 mm-gapped cuvette (BioRad Laboratories Inc., Hercules, CA). The cells were transformed by electroporation using Gene Pulser II (BioRad) at 2.5 kV, 25 μF, and 200 Ω. After electroporation, 1 mL of SOC medium (Invitrogen, Carlsbad, CA) was immediately added to the cell suspension, and the culture was incubated at 37 °C for 1 h. One hundred microliters of the cell suspension was plated on TSAY containing 50 μg mL−1 of kanamycin (Nacarai Tesque, Kyoto, Japan). Four hundred and eighty-six colonies grown on selection plates were transferred into TSBY containing 50 μg mL−1 of kanamycin for screening mutants deficient in exopolysaccharide production.

Screening for mutants deficient in exopolysaccharide production and meshwork-like structures

The viscosity of spent culture media of 486 mutants was measured using a rotary viscometer (Tokimec Inc.) as described above. Mutants showing lower viscosity than that of the parent strain YS-11 were further investigated by means of SEM to observe cell surface-associated structures as described previously. Mutants that had completely lost the meshwork-like structures around cells were selected as putative knockout mutants for genes involved in the formation of biofilm-like structures.

Detection of transposon insertion by Southern hybridization

Southern hybridization was carried out to confirm a single insertion of transposon on genomic DNA. The genomic DNA from a mutant strain without exopolysaccharide production was purified using the GNOME Kit (Qbiogene Inc., Morgan Irvine, CA) and digested with a restriction enzyme PstI (Takara Bio, Ohtsu, Japan). The DNA fragments were electrophoresed on a 0.8% SeaKem agarose gel (Takara Bio), transferred to a positively charged nylon membrane (Hybond-N+, Amersham Biosciences Corp., Piscataway, NJ), and fixed on the membrane by UV light irradiation (HL-2000 HybriLinker, UVP Inc., Upland, CA). To detect an insertion of EZ-Tn5 Tnp, a digoxigenin (DIG)-labeled probe designed from the sequence of EZ-Tn5 Tnp was generated using the PCR DIG probe synthesis Kit (Roche Applied Science, Mannheim, Germany) with a primer pair (Table 1) to amplify a kanamycin-resistant gene in EZ-Tn5 Tnp (EZ-Tn5 Tnp sequence is available at http://www.epibio.com/pdftechlit/techlit_eztn.asp). The membrane was prehybridized (30 min, 65 °C) in a hybridization solution (DIG Easy Hyb Granules, Roche Applied Science) and subsequently hybridized overnight at 65 °C with 2 μL mL−1 of DIG-labeled probe in a hybridization solution. The detection of DIG-labeled probes was carried out according to the manufacturer's instruction in a DIG Luminescent Detection Kit (Roche Applied Science).

Table 1.   Primers used in this study
ExperimentsPrimerSequence*
  • *

    Restriction sites are underlined.

  • First PCR primers were, respectively, combined with one of the DNA walking annealing control primers provided by a kit (DW-ACP1: 5′-ACP-AGGTC-3′; DW-ACP2: 5′-ACP-TGGTC-3′; DW-ACP3: 5′-ACP-GGGTC-3′; DW-ACP4: 5′-ACP-CGGTC-3′). Nested PCR primers were combined with a DW-ACP for nested PCR (DW-ACPN: 5′-ACPN-GGTC-3′), respectively. Second nested PCR primers were combined with a universal primer (5′-TCACAGAAG TATGCCAAGCGA-3′).

DIG-labeled probeKAN2-15′-ATCAGGTGCGACAATCTATC-3′
KAN2-3R5′-ACCGAGGCAGTTCCATAG-3′
DNA walking
First PCREZTN-R5′-CTCAAAATCTCTGATGTTACATTGC-3′
EZTN-F5′-GGTTGATGAGAGCTTTGTTGTAGGT-3′
Nested PCRKAN2-15′-ATCAGGTGCGACAATCTATC-3′
KAN2-3R5′-ACCGAGGCAGTTCCATAG-3′
Second nested PCRKAN-2FP15′-ACCTACAACAAAGCTCTCATCAACC-3′
KAN-2RP15′-GCAATGTAACATCAGAGATTTTGAG-3′
Wzt complementationwzt-EcoF5′-ACGAATTCTATGAAAAACTCTATCAAGCTG-3′
wzt-PstR5′-ACGAATTCGTTTTGATGGGTAGAAAGATGG-3′

Flanking regions of an insertion of EZ-Tn5 Tnp

Alignments of flanking regions of the inserted EZ-Tn5 Tnp were analyzed using a DNA Walking SpeedUp Premix Kit (Seegene Inc., Seoul, Korea) according to the instruction of the kit. Transposon-specific primers for the first PCR (EZTN-F and EZTN-R, Table 1) were designed from a kanamycin-resistant gene in EZ-Tn5 Tnp and these were combined with DNA walking annealing control primers (DW-ACP1, 5′-ACP-AGGTC-3′; DW-ACP2, 5′-ACP-TG GTC-3′; DW-ACP3, 5′-ACP-GGGTC-3′; DW-ACP4, 5′-ACP-CGGTC-3′) provided by the kit (Seegene Inc.) for determination of the flanking regions of the insertion. Genomic DNA of mutants were prepared as described above. The first PCR reaction was performed with eight different primer pairs in which one of the DW-ACPs was combined with EZTN-F or EZTN-R. PCR amplification was carried out at 94 °C for 5 min, 42 °C for 1 min, 72 °C for 2 min, and then 30 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 1 min, followed by 72 °C for 7 min. The first nested PCR was performed using primer pairs of EZ-Tn5 Tnp-specific nested primers KAN2-1or KAN2-3R (Table 1) and a DW-ACP for nested PCR (DW-ACPN: 5′-ACPN-GGTC-3′) provided by the kit (Seegene Inc.). Two microliters of the first PCR product was used as template DNA. PCR amplification was carried out at 94 °C for 5 min, and then 35 cycles of 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 1 min, followed by 72 °C for 7 min. The second nested PCR was performed using primer pairs of EZ-Tn5 Tnp-specific second nested primers (KAN-2FP1 or KAN-2RP1 provided by the EZ-Tn5 Tnp Kit (Epicentre Biotechnologies, Table 1) and a universal primer (5′-TCACAGAAGTATGCCAAGCGA-3′) provided by the kit (Seegene Inc.). One microliter of the first nested PCR product was used as template DNA. Conditions for PCR were as follows: 94 °C for 5 min, then 35 cycles at 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 1 min, followed by 72 °C for 7 min. The PCR products were electrophoresed, isolated, and cloned using the TOPO TA Cloning system (Invitrogen). Plasmids containing the PCR products were purified using the QIAprep Spin MiniPrep Kit (Qiagen Science, MD). The PCR products were then sequenced using the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) with a pair of M13 primers. The DNA sequences obtained were converted into amino acid sequences using genetyx ver. 7.0 software (Genetyx Co. Ltd, Tokyo, Japan). Homology searches of amino acid sequences were performed using the fasta algorithm in the DDBJ (Mishima, Japan). The sequence of the flanking regions of the EZ-Tn5 Tnp insertion has been submitted to the DDBJ nucleotide sequence database (DDBJ accession: AB377402).

Complementation of the disrupted gene to a mutant deficient in exopolysaccharide production

Among 486 mutants, we found only one mutant (strain 455) that had lost the ability to produce exopolysaccharide and form meshwork-like structures. The sequencing analysis of the flanking regions of the transposon insertion revealed that the transposon was inserted into an ORF highly homologous to wzt in the per cluster of Y. enterocolitica serotype O:9 (Lubeck et al., 2003; Skurnik, 2003; Jacobsen et al., 2005). We further performed a complementation study to determine whether the wild-type gene (wztYS-11) was associated with the viscous material production and could restore this phenotype in strain 455. The wztYS-11 was introduced into strain 455, and the changes in the phenotype were observed by SEM. The fragment including the wztYS-11 ORF was amplified by PCR using the primers wzt-EcoF and wzt-PstR (Table 1). An EcoRI site or a PstI site (Table 1, underlined) was introduced into the 5′ end of the PCR product. The reaction mixture contained 10 ng μL−1 genome DNA of strain YS-11, 1 × PCR buffer, 0.2 mM dNTPs, 0.5 μM each primer, and 25 mU μL−1 KOD dash DNA polymerase (Toyobo, Osaka, Japan), and sterile-distilled water was added to the mixture to a final volume of 50 μL. The reaction conditions were as follows: 94 °C for 6 min, 35 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, and 72 °C for 2 min with a PCR thermal cycler (Takara Bio). The PCR product purified with the QIAquick gel extraction kit (Qiagen) was digested with EcoRI and PstI (Takara Bio), and ligated to the plasmid vector pSTV28 (Takara Bio), which was predigested with the same combination of restriction enzymes. Ligation was performed using a DNA ligation kit ver. 2.1 (Takara Bio) according to the manufacturer's directions. Escherichia coli DH5α (Invitrogen) was transformed with this ligation solution. The constructed plasmid, named pWZT, was purified from a colony grown on TSAY containing 20 μg mL−1 of chloramphenicol. Ten nanograms of constructed plasmid was added to 50 μL of the competent cell of strain 455, and transformation was carried out as described above. Measurement of the viscosity of spent culture media and SEM observation for the presence of meshwork-like surface structures were carried out on the recombinants grown on the TSAY containing 50 μg mL−1 of kanamycin and 20 μg mL−1 of chloramphenicol. The wztYS-11 on the pWZT was fused with the α-peptidase gene on pSTV28 so that the viscosity and the cell surface-associated phenotype were examined under culture conditions with or without 1 mM isopropyl-β-d(−)-thiogalactopyranoside (IPTG; Wako Pure Chemical Industries, Osaka, Japan). Strain 455 with pSTV28 and E. coli DH5α with pWZT were used as controls. The bacterial strains and plasmids used in this study are listed in Table 2.

Table 2.   Bacterial strains and plasmids used in this study
Strain or plasmidRelevant descriptionRelevant phenotype or source
E. coli DH5αlacZΔM15 recA1Invitrogen
E. hermannii
 YS-11Clinical isolate from persistent apical periodontitisMeshwork-positive
 455wzt178∷Tn5, generated from YS-11Meshwork-negative
 455-LMStrain 455 transformed with pWZTMeshwork-positive
 455-pSTV28Strain 455 tranformed with pSTV28Meshwork-negative
 ATCC 33650Reference strainMeshwork-negative
pSTV28Expression vectorTakara
pWZTpSTV28 carrying the wild-type wzt of E. hermanniiThis study
pCR2.1-TOPOCloning vectorInvitrogen

Animal studies

Escherichia hermannii strains YS-11 and 455-LM with meshwork-like structures were compared with those of strains 455 and ATCC33650 that lacked this phenotype for the ability to induce abscess formation in mice. Bacterial strains were cultured in TSBY for 12 h (early stationary phase). Five hundred microliters of bacterial suspensions (107–109 CFU mL−1) were injected subcutaneously into the inguen of each BALB/c mouse (male, 4 weeks; three mice per strain). Changes in abscess lesions were recorded photographically using a camera (Nikon FIII, Nikon, Japan) set at a fixed magnification for five consecutive days.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Viscosity of spent culture media and cell surface-associated structures of YS-11 and ATCC33650

Stock cultures of YS-11 were inoculated into TSBY and grown for 48 h. The viscosities of the spent culture media were measured using a rotary viscometer. All the YS-11 stocks tested produced materials in vitro that were highly viscous as compared with the control TSB medium. In contrast, the viscosity of the spent culture medium obtained from ATCC33650 was similar to that of the control TSBY medium (Fig. 1a). SEM observations on the cell surfaces of these strains revealed that YS-11 had meshwork-like structures surrounding the cells (Fig. 1b), but ATCC33650 lacked this phenotype (Fig. 1c).

image

Figure 1.  (a) Viscosities of spent culture media obtained from Escherichia hermannii strains YS-11 (a clinical isolate from a persistent apical periodontitis lesion) and ATCC33650 (reference strain). TSBY, control medium without bacterial inoculation. *P<0.01. (b and c) SEMs showing surface structures of E. hermannii strains YS-11 (wild type) and 33650 (a reference strain for E. hermannii). YS-11 had fibrillar structures around cells (b), but ATCC33650 (c) did not. Scale bars=2 μm.

Download figure to PowerPoint

Chemical composition of the viscous materials isolated from YS-11 culture supernatants

Chemical analyses showed that the isolated materials primarily consisted of neutral sugars, small amounts of uronic acid, and amino sugars, with mannose constituting 78.4% of the polysaccharides (Table 3). Lipopolysaccharide activity in the purified viscous materials was 0.33±0.08 EU mg−1.

Table 3.   Amount of neutral sugar, neutral sugar components, uronic acid and amino-sugar in the viscous material isolated from Escherichia hermannii YS-11
SugarAmount (μg mg−1)
Neutral sugar535.7
 Mannose467.7
 Glucose23.4
 Fructose17.8
 Galactose10.5
 Arabinose7.1
 Xylose4.8
 Rhamnose4.4
Uronic acid33.8
Amino-sugar26.3

Construction of a mutant led to a complete loss of the meshwork-like structures

We constructed a mutant that lacked the ability to produce exopolysaccharide in the culture supernatant and to form meshwork-like structures around cell surfaces by random insertion of EZ-Tn5 Tnp to chromosomal DNA of YS-11. Among 486 colonies grown on TSAY-Km, only one strain (strain 455) showed low viscosity in its culture medium as a control level (Fig. 2) and cell surfaces without meshwork-like structures (Fig. 3a). Southern hybridization indicated that strain 455 had an insertion of EZ-Tn5 Tnp (data not shown). Sequencing analysis by DNA walking showed that the transposon in strain 455 was inserted into an ORF that was highly homologus to wzt. This gene encodes the ATP-binding protein of the ABC transporter system in the O-antigen biosynthesis gene cluster of Y. enterocolitica serotype O:9 (Lubeck et al., 2003; Skurnik, 2003) (Fig. 4). The upper region of wzt ORF contains homologues of Erwinia chrysanthemi manB (Touze et al., 2004), gmd, per, wzm of Aeromonas hydrophila (Seshadri et al., 2006) or Y. enterocolitica serotype O:9 (Lubeck et al., 2003; Skurnik, 2003), and wbcT of Y. enterocolitica serotype O:9 (Lubeck et al., 2003; Skurnik, 2003). The flanking regions of the transposon insertion are depicted in Fig. 4.

image

Figure 2.  Viscosities of spent culture media obtained from Escherichia hermannii strains 455 (wzt transposon insertion mutant generated from YS-11) and 455-LM (strain 455 transformed with pWZT). IPTG(+), with 1 mM IPTG; IPTG(−), without IPTG; TSBY, control medium without bacterial inoculation. *P<0.01.

Download figure to PowerPoint

image

Figure 3.  SEMs showing surface structures of Escherichia hermannii (a) strain 455 (wzt mutant generated from YS-11), (b) 455-LM (strain 455 transformed with pWZT) without IPTG, and (c) 455-LM with 1 mM IPTG. Note the very dense fibrillar structures on 455-LM (b and c). Scale bars=2 μm.

Download figure to PowerPoint

image

Figure 4.  Schematic depiction of flanking regions of EZ-Tn5 transposon insertion on Escherichia hermannii strain 455.

Download figure to PowerPoint

Complementation of wzt to a mutant deficient in exopolysaccharide production

To further investigate how wztYS-11 was involved in viscous material production, we constructed a plasmid pWZT carrying the wztYS-11 ORF in which wzt was fused with the lacZα-peptide gene on the pSTV28 to complement the mutant strain lacking this phenotype. Plasmid pWZT was introduced into strain 455. The resultant recombinant, designated as strain 455-LM, was capable of producing extracellular materials of higher viscosity (Fig. 2) and cell surface-associated meshwork-like materials as revealed by SEM (Fig. 3b) than those of strain 455. IPTG induction augmented both the viscosity of the extracellular viscous material and the abundance of meshwork-like structures around cells (Figs 2 and 3c). Control strains, strains 455-pSTV28 and E. coli DH5α-pWZT, exhibited any changes of the above-described phenotypes (data not shown).

Abscess induction in mice

The ability to induce abscess formation in mice by E. hermannii strains YS-11 (a clinical isolate with the per cluster), 455 (wzt transposon insertion mutant generated from YS-11), 455-LM (strain 455 transformed with pWZT), and ATCC33650 (reference strain without the per cluster) was compared. Strains YS-11 and 455-LM induced abscess lesions in mice at 107 CFU mL−1. In contrast, strains 455 and ATCC33650 required 109 CFU mL−1 to induce abscess lesions in mice (Fig. 5).

image

Figure 5.  Ability to induce abscess formation in mice of Escherichia hermannii strains YS-11 (a clinical isolate with the per cluster), 455 (wzt transposon insertion mutant generated from YS-11), 455-LM (strain 455 transformed with pWZT), and ATCC33650 (reference strain without the per cluster). Strains YS-11 and 455-LM induced abscess lesions in mice at 107 CFU mL−1. In contrast, strains 455 and ATCC33650 needed 109 CFU mL−1 to induce apparent abscess lesions in mice. Data are representative of two independent experiments.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

In this study, we described some of the pathogenic properties of a clinical strain of E. hermannii that was isolated from a persistent apical periodontitis (Chavez de Paz, 2007; Yamane et al., 2009) lesion. Apical periodontitis is a relatively common inflammatory disease in dentistry, and a wide variety of bacterial genera including enteric bacteria have been implicated as putative pathogens (Fukushima et al., 1990; Sundqvist et al., 1998; Peciuliene et al., 2001). The ability to form biofilms has recently been considered to be crucial for microorganisms that are present in a root canal to resist the intraroot canal procedures of disinfection, to occupy apical foramina of teeth, and to cause persistent chronic inflammatory lesions (Fukushima et al., 1990; Chavez de Paz, 2007). Although bacteria belong to the family Enterobacteriaceae, such as E. coli, Proteus spp., and Klebsiella pneumoniae are occasionally isolated from chronic and asymptomatic lesions (Yoshida et al., 1987; Peciuliene et al., 2001), the association of E. hermannii with apical periodontitis has not been reported before.

Exopolysaccharide production and the presence of cell surface-associated meshwork-like structures are some of the common features associated with biofilm-forming bacteria (Kobayashi, 1995; Zogaj et al., 2003; Yamanaka et al., 2009). Strain YS-11 produced an abundance of mannose-rich exopolysaccharides and cell surface-associated fibrillar structures. Some of the phenotypes described here for strain YS-11 are similar to those of Pseudomonas aeruginosa, a prototype biofilm-forming bacterium (Kobayashi, 1995; Yasuda et al., 1999), E. coli (Prigent-Combaret et al., 2000; Uhlich et al., 2006), Salmonella (Anriany et al., 2001; Jain & Chen, 2006), and V. cholerae (Wai et al., 1998). Although these bacteria produce different exopolysaccharides with different chemical natures, for example alginate or galactose and mannose-rich exopolysaccharide Psl for P. aeruginosa biofilms (Ryder et al., 2007), colanic acid for E. coli K-12 (Prigent-Combaret et al., 2000), and cellulose for Salmonella (Zogaj et al., 2003), they all form cell surface-associated dense meshwork-like structures. In this study, we found that the wzt mutation in the perosamine synthesis gene cluster of YS-11 prevented the production of the meshwork-like structures by this organism. As described above, perosamine is the common O-chain of lipopolysaccharides in several different bacteria (Perry & Bundle, 1990; Rice et al., 1992; Godfroid et al., 1998; Reeves & Wang, 2002; Munoz et al., 2005). Among the bacteria possessing the perosamine biosynthesis system, E. coli O157:H7 (Uhlich et al., 2006) and V. cholerae O1 (Wai et al., 1998) resemble E. hermannii YS-11 with regard to the presence and the morphology of cell surface structures. In contrast, ATCC33650, known to lack perosamine (Perry & Bundle, 1990), did not exhibit this phenotype. A recent study (Sheng et al., 2008) showed that the deletion of per in E. coli O157:H7 resulted in a mutant lacking the O antigen with a concomitant nonmotile, autoaggregative phenotype. The liquid cultures of this mutant also showed more rapid sedimentation than that of the parent strain. When we compared the turbidity of spent culture media obtained from strains YS-11 (wild type), 455 (wzt-deleted mutant), 455-LM (complemented strain), and ATCC33650 (per negative) cultures, both strains 455 and ATCC33650 cells showed rapid sedimentation in the medium (data not shown).

Because strains YS-11 and 455-LM induced greater abscess formation in mice than did strains 455 and ATCC33650, it is likely that the biofilm-like structures as described above for these strains might be important for the pathogenicity of E. hermannii. However, it is important to note that the data presented were derived from the study of one clinical isolate; therefore, the results might not be representative of the overall pathogenic potential of this organism. As for future studies, we will examine other strains of E. hermannii for the presence of the per cluster. More thorough investigations are also needed to determine the role of this gene cluster in biofilm formation by this organism, although the data obtained from this study strongly suggest that the wzt is involved in the exopolysaccharide production.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

We are grateful to Mr Hideaki Hori (the Institute of Dental Research, Osaka Dental University) for his excellent assistance with the electron microscopy. A part of this research was performed at the Institute of Dental Research, Osaka Dental University. This study was supported in part by the Osaka Dental University Research Fund (A05-09) and Osaka Dental University Joint Research Funds (B08-01).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  • Adam O, Vercellone A, Paul F, Monsan PF & Puzo G (1995) A nondegradative route for the removal of endotoxin from exopolysaccharides. Anal Biochem 225: 321327.
  • Anriany YA, Weiner RM, Johnson JA, De Rezende CE & Joseph SW (2001) Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype. Appl Environ Microb 67: 40484056.
  • Beauchef-Havard A, Arlet G, Gautier V, Labia R, Grimont P & Philippon A (2003) Molecular and biochemical characterization of a novel class A β-lactamase (HER-1) from Escherichia hermannii. Antimicrob Agents Ch 47: 26692673.
  • Brenner DJ, Davis BR, Steigerwalt AG et al. (1982) Atypical biogroups of Escherichia coli found in clinical specimens and description of Escherichia hermannii sp. nov. J Clin Microbiol 15: 703713.
  • Campbell JH & Pappenheimer AM (1966) Quantitative studies of the specificity of anti-pneumococcal polysaccharide antibodies, types 3 and 8. II. Inhibition of precipitin reactions with oligosaccharides isolated from hydrolysates of S3 and S8. Immunochemistry 3: 213222.
  • Chaudhury A, Nath G, Tikoo A & Sanyal SC (1999) Enteropathogenicity and antimicrobial susceptibility of new Escherichia spp. J Diarrhoeal Dis Res 17: 8587.
  • Chavez de Paz LE (2007) Redefining the persistent infection in root canals: possible role of biofilm communities. J Endodont 33: 652662.
  • Dahl KM, Barry J & DeBiasi RL (2002) Escherichia hermannii infection of a cephalohematoma: case report, review of the literature, and description of a novel invasive pathogen. Clin Infect Dis 35: e96e98.
  • Davidson AL & Chen J (2004) ATP-binding cassette transporters in bacteria. Annu Rev Biochem 73: 241268.
  • Fitoussi F, Arlet G, Grimont PAD, Lagrange P & Philippon A (1995) Escherichia hermannii: susceptibility pattern to β-lactams and production of β-lactamase. J Antimicrob Chemoth 36: 537543.
  • Fukushima H, Yamamoto K, Hirohata K, Sagawa H, Leung KP & Walker C (1990) Localization and identification of root canal bacteria in clinically asymptomatic periapical pathosis. J Endodont 16: 534538.
  • Ginsberg HG & Daum RS (1987) Escherichia hermannii sepsis with duodenal perforation in a neonate. Pediatr Infect Dis J 6: 300302.
  • Godfroid F, Taminiau B, Danese I et al. (1998) Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages. Infect Immun 66: 54855493.
  • Hernandez A, Mellado RP & Martinez JL (1998) Metal accumulation and vanadium-induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae. Appl Environ Microb 64: 43174320.
  • Jacobsen NR, Bogdanovich T, Skurnik M, Lubeck PS, Ahrens P & Hoorfar J (2005) A real-time PCR assay for the specific identification of serotype O:9 of Yersinia enterocolitica. J Microbiol Meth 63: 151156.
  • Jain S & Chen J (2006) Antibiotic resistance profiles and cell surface components of Salmonellae. J Food Protect 69: 10171023.
  • Kobayashi H (1995) Airway biofilm disease: clinical manifestations and therapeutic possibilities using macrolides. J Infect Chemother 1: 115.
  • Lubeck PS, Hoorfar J, Ahrens P & Skurnik M (2003) Cloning and characterization of the Yersinia enterocolitica serotype O:9 lipopolysaccharide O-antigen gene cluster. Adv Exp Med Biol 529: 207209.
  • Munoz PM, Marin CM, Monreal D et al. (2005) Efficacy of several serological tests and antigens for diagnosis of bovine Brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9. Clin Diagn Lab Immun 12: 141151.
  • Peciuliene V, Reynaud AH, Balciuniene I & Haapasalo M (2001) Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J 34: 429434.
  • Perry MB & Bundle DR (1990) Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus. Infect Immun 58: 13911395.
  • Pien FD, Shrum S, Swenson JM, Hill BC, Thornsberry C & Farmer JJ III (1985) Colonization of human wounds by Escherichia vulneris and Escherichia hermannii. J Clin Microbiol 22: 283285.
  • Poulou A, Dimitroulia E, Markou F & Tsakris A (2008) Escherichia hermannii as the sole isolate from a patient with purulent conjunctivitis. J Clin Microbiol 46: 38483849.
  • Prigent-Combaret C, Prensier G, Le Thi TT, Vidal O, Lejeune P & Dorel C (2000) Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid. Environ Microbiol 2: 450464.
  • Reeves PP & Wang L (2002) Genomic organization of LPS-specific loci. Curr Top Microbiol 264: 109135.
  • Rice EW, Sowers EG, Johnson CH, Dunnigan ME, Strockbine NA & Edberg SC (1992) Serological cross-reactions between Escherichia coli O157 and other species of the genus Escherichia. J Clin Microbiol 30: 13151316.
  • Ryder C, Byrd M & Wozniak DJ (2007) Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr Opin Microbiol 10: 644648.
  • Seshadri R, Joseph SW, Chopra AK et al. (2006) Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades. J Bacteriol 188: 82728282.
  • Sheng H, Lim JY, Watkins MK, Minnich SA & Hovde CJ (2008) Characterization of an Escherichia coli O157:H7 O-antigen deletion mutant and effect of the deletion on bacterial persistence in the mouse intestine and colonization at the bovine terminal rectal mucosa. Appl Environ Microb 74: 50155022.
  • Skurnik M (2003) Molecular genetics, biochemistry and biological role of Yersinia lipopolysaccharide. Adv Exp Med Biol 529: 187197.
  • Sundqvist G, Figdor D, Persson S & Sjogren U (1998) Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med O 85: 8693.
  • Touze T, Goude R, Georgeault S, Blanco C & Bonnassie S (2004) Erwinia chrysanthemi O antigen is required for betaine osmoprotection in high-salt media. J Bacteriol 186: 55475550.
  • Uhlich GA, Cooke PH & Solomon EB (2006) Analyses of the red-dry-rough phenotype of an Escherichia coli O157:H7 strain and its role in biofilm formation and resistance to antibacterial agents. Appl Environ Microb 72: 25642572.
  • Wai SN, Mizunoe Y, Takade A, Kawabata S-I & Yoshida S-I (1998) Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation. Appl Environ Microb 64: 36483655.
  • Yamanaka T, Furukawa T, Matsumoto-Mashimo C et al. (2009) Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17. BMC Microbiol 9: 11.
  • Yamane K, Ogawa K, Yoshida M et al. (2009) Identification and characterization of clinically isolated biofilm-forming gram-positive rods from teeth associated with persistent apical periodontitis. J Endodont 35: 347352.
  • Yasuda H, Koga T & Fukuoka T (1999) In vitro and in vivo models of bacterial biofilms. Method Enzymol 310: 577595.
  • Yoshida M, Fukushima H, Yamamoto K, Ogawa K, Toda T & Sagawa H (1987) Correlation between clinical symptoms and microorganisms isolated from root canals of teeth with periapical pathosis. J Endodont 13: 2428.
  • Zogaj X, Bokranz W, Nimtz M & Romling U (2003) Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract. Infect Immun 71: 41514158.