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Applied and Environmental Microbiology, October 2007, p. 6321-6325, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00606-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Use of the tna Operon as a New Molecular Target for Escherichia coli Detection{triangledown}

Camilla Bernasconi,1,{dagger} Giorgio Volponi,2,{dagger} Claudia Picozzi,2* and Roberto Foschino2

European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Ispra (VA), Italy,1 DiSTAM, Università degli Studi di Milano, Milano, Italy2

Received 16 March 2007/ Accepted 2 August 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 Nucleotide sequence accession...
 REFERENCES
 
A quantitative real-time PCR targeting the tnaA gene was studied to detect Escherichia coli and distinguish E. coli from Shigella spp. These microorganisms revealed high similarity in the molecular organization of the tna operon.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
Nucleotide sequence accession...
 REFERENCES
 
Council Directives 2006/7/EC (1) and 98/83/EC (2) on water quality and Commission Regulation 2073/2005 (7) on microbiological criteria for foodstuffs require the determination of Escherichia coli to assure hygiene control and consumer safety. Nowadays, gene probe technology provides rapid and highly sensitive techniques for the specific detection of pathogenic microorganisms (6, 8, 11, 15). E. coli is able to use tryptophan as a carbon source, and this phenotypic trait is normally employed as a test for identification in conventional culture-based techniques. The tryptophanase operon (tna) is a 3,049-bp region consisting of two major structural genes: tnaA (1,431 bp), coding for the tryptophanase that catalyzes the degradation of L-tryptophan to indole, pyruvate, and ammonia; and tnaB (1,248 bp), coding for a tryptophan permease (21). tnaA is preceded by a transcribed regulatory leader region containing a short open reading frame, tnaL, specifying a 25-residue leader peptide. In between there is a 205-bp spacer region that contains several transcription pause sites. Moreover, in silico analysis of complete genomes of eight E. coli strains available in GenBank confirmed that the tna operon is present in single copy, ranking it as a promising molecular biomarker for quantification of the target organism. The aim of this study was to develop a quantitative real-time PCR (Q-PCR) assay to detect pathogenic and nonpathogenic strains of E. coli, differentiating them from Shigella spp., which are closely related bacteria. Actually the two taxa are often difficult to distinguish by phenotypic traits, while, at genome level, some authors consider them as belonging to the same species (4, 5, 14). In the current study the molecular basis of the indole phenotype in Shigella and Escherichia species was examined by an investigation of the molecular organization of the tna operon. One-hundred eighteen E. coli strains and 100 non-E. coli strains representative of other enteric and environmental species, collected from different sources and international collections, were studied (Table 1). All the new isolates were identified by the Vitek system (bioMérieux, Marcy l'Etoile, France) and/or through the sequencing of the 16S rRNA gene operon. After a preliminary screening, the gene encoding tryptophanase (tnaA) was chosen as target for the design of E. coli-specific PCR primers and of a TaqMan MGB probe by comparing relative gene sequences of E. coli K-12 with those of Shigella flexneri 2a strain 2457T, E. coli O157:H7 EDL 933, and E. coli CFT073 (Table 2). The rationale behind this selection was to identify genomic regions able to discriminate the two species, since published protocols are not suitable for this purpose (3, 9).


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  		TABLE 1. Strains used in this work

 

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  		TABLE 2. Primers and probe designed in this work and relative amplification conditions

 
Real-time PCR amplification was performed in a 25-µl volume containing 1x TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 1 µM of primers, 200 nM of probe, and 100 ng of DNA template. Tests were performed in triplicate in an ABI Prism 7900 thermal cycler (Applied Biosystems), using MicroAmp optical tubes and caps (Applied Biosystems). Amplification conditions are reported in Table 2. One hundred five out of 118 E. coli strains (89%) gave positive signal in the Q-PCR test, while no amplification signal was observed in 98 out of 100 non-E. coli strains (98%). In particular, all Shigella sp. isolates were negative. According to ISO/FDS 16140:2003 (12), the new assay showed an accuracy of 93.1% and a specificity of 98.0%. Thirteen E. coli strains, 12 with an indole-positive phenotype, did not produce the expected fragment: CD3, DSM 682, E01, E05, E16, E22, E81 (indole negative), EC52, ED172 (O103:H2), ED226 (O113:H21), EF1 (O111:H–), EscCol1, and EscCol2. On the other hand, Escherichia albertii DSM 17582T (indole negative) and Escherichia fergusonii DSM 13698T (indole positive) strains gave a false-positive amplification signal. Then, the tna operon was entirely sequenced through a gene walking analysis using primers tnaOP_F and tnaOP_R, designed on the 5' region of tnaL and on the 3' region of tnaB of E. coli K-12, respectively (Table 2). The same survey was extended to Shigella boydii DSM 7532T, Shigella flexneri DSM 4782T, and Shigella sonnei ATCC 29930T, SHIson1, and PO2. PCR was performed as described above, and the amplification cycles are reported in Table 2. Both strands of the amplification products of the tna operon were sequenced, analyzed by the BLAST 2 sequence program, and deposited in the GenBank database. A multiple-alignment distance, determined by the unweighted pair group method using arithmetic averages was used to draw a tree based on tna operon sequences by the Kodon software (Applied Maths, Kortrijk, Belgium). S. sonnei strains did not amplify the tna operon. E. coli E81, E. albertii DSM 17582T, and S. boydii DSM 7532T isolates amplified regions of 4,387 bp, 3,827 bp, and 2,814 bp, respectively. The other strains, including E. fergusonii DSM 13698T and S. flexneri DSM 4782T, amplified a region of 3,065 bp as expected. The dendrogram obtained by the alignment of tna operon sequences allows discrimination of two major clusters of nucleotide distance at the 72.1% level (Fig. 1). Cluster A includes all the Escherichia spp. and four strains of Shigella spp. without insertion sequences (S. boydii B12 and S. flexneri 2a strain 2457T, 5 strain 8401, and 2a strain 301); cluster B groups all the other Shigella sp. strains presenting insertion sequence (IS) elements and showing an indole-negative phenotype. E. coli K-12 W3110 has two complete IS5 elements (1,195 bp): one in the intragenic tnaL-tnaA region and the other in the tnaB gene (Fig. 2). Isolate E81, from a healthy individual, had a tnaA gene that was interrupted by a 1,329-bp IS, which showed a 100% homology with IS10 present in Salmonella enterica serovar Typhimurium (17). E. albertii DSM 17582T has an IS of 768 bp (IS1A) in the spacing region between tnaL and tnaA; in addition, two nucleotide deletions (T1785 and C2587) and one insertion (A1718) with respect to E. coli K-12 were detected. As a consequence, these strains are unable to produce a functional enzymatic system for tryptophan degradation. In E. coli ATCC 11775T, E. coli UTI89, avian pathogenic E. coli O1, E. coli 536, E. coli CFT073, and E. fergusonii DSM 13698T (cluster A1) the tna operon has the same organization as in E. coli K-12 but with a single nucleotide insertion (A1718) in the region between tnaA and tnaB. Since this insertion falls in a noncoding region, open reading frames are not affected and strains are phenotypically indole positive. With the exception of DSM 10650 and two O157:H7 isolates (EDL933 and strain Sakai), all the E. coli strains of cluster A2 present, compared to E. coli K-12, three point mutations in the region from which primer tnaA_F was designed: G472 to A, G475 to C, and G788 to A. Moreover, apart from strains ED226 (O113:H21), ED172 (O103:H2), and EF1 (O111:H–), they also present two point mutations in correspondence to primer tnaA_R: C580 to T and C583 to T. All these mutations take place on the third base of the codon, and, since the amino acid does not change, they do not affect the translation into a functional tryptophanase. However, they decrease the annealing efficiency during PCR amplification, giving rise to a false-negative signal. It is noteworthy that the same point mutations are present in Shigella strains grouped in this cluster. Shigella boydii B12 has an indole-negative phenotype since, as reported by Rezwan et al. (20), it presents an IS before the region we have investigated that disrupts the expression of the tna operon. Cluster B groups Shigella spp. whose tna operons are affected by insertion elements causing the indole-negative phenotype. In particular, S. boydii strains in cluster B1 show one partial 192-bp IS1 sequence followed by a full IS1 sequence; these sequences determine a deletion of 49 bp, including 21 bp of tnaL, and they present the same point mutations in tnaA primers annealing regions already described for E. coli strains of cluster A2. S. boydii and S. flexneri strains of cluster B2 displayed instead a full IS1 of 768 bp starting from base 55 of tnaL; the insertion was followed by the deletion of the complete 205-bp interspace region between tnaL and tnaA and of the first 777 bp in the 5' start sequence of the tnaA gene; therefore, 235 bp is missing. Shigella dysenteriae strains (cluster B3) presented the same 768-bp IS, IS1, at base 55 of tnaL of cluster B2, but without any deletion. Moreover, strain D3 showed the presence of another IS1 located in an opposite orientation on the tnaA gene, giving rise to a lower nucleotide homology (71.0%) compared to other strains (>99.9%) of the same cluster. Furthermore, these isolates presented, in the regions from which primers were drawn, the same point mutations detected on cluster B1 and A2 strains. The high frequency of IS elements is known to mediate various genetic rearrangements, including inversions and deletions, that could play an important role in the evolution of the taxa. Besides, the occurrence of gene transfer by conjugation, transduction, and formation of recombinants between S. flexneri and E. coli, particularly for pathogenic serotypes, has already been demonstrated (16, 18). IS elements have often been associated with negative phenotypes since they can disrupt the functionality of the genes (20); in our study this trait was highlighted through the sequence analysis of the E81 strain, the sole E. coli isolate with a negative indole phenotype. The other E. coli strains that do not gave amplification signals in this Q-PCR assay reveal the same point mutations and the same organization of the operon as some S. boydii and S. flexnerii strains, corroborating the hypothesis that the Shigella pathotype arose from E. coli ancestors (7, 19). Anyway, it is not possible to find other regions in the tna operon that could allow the discrimination between the species that we considered in this work. Although E. fergusonii and E. albertii gave a false-positive signal, this fact does not invalidate the test since they have the same habitat as E. coli (10). Finally, this PCR assay can be employed as a rapid preliminary tool, even if it should be integrated with phenotypic results.


Figure 1
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  		FIG. 1. Tree obtained by the unweighted-pair group method using arithmetic averages (UPGMA) for tna operon sequences of Escherichia and Shigella species. Bootstrap values are indicated on each node of the tree (1,000 pseudoreplicates). *, sequences obtained in this work; nd, not determined.

 

Figure 2
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  		FIG. 2. Comparison of gene organizations of tna operons from Escherichia and Shigella species.

 

   Nucleotide sequence accession numbers.
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 ABSTRACT
 INTRODUCTION
 Nucleotide sequence accession...
REFERENCES
 
Nucleotide sequences obtained in this work have been deposited in the GenBank database under accession numbers EF445878 to EF445895.


   FOOTNOTES
 
* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, via Celoria 2, 20133 Milano, Italy. Phone: 39 02 5031 9174. Fax: 39 02 5031 9191. E-mail: Claudia.Picozzi{at}unimi.it Back

{triangledown} Published ahead of print on 10 August 2007. Back

{dagger} Both the authors contributed to the work at the same level. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 Nucleotide sequence accession...
 REFERENCES
 

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Applied and Environmental Microbiology, October 2007, p. 6321-6325, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00606-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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