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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 4  |  Page : 528-532
 

Molecular characterisation of enteroinvasive Escherichia coli O136:K78 isolates from patients of a diarrhoea outbreak in China


Department of Microbiology, Nanchang Center for Disease Control and Prevention, Nanchang, China

Date of Submission23-May-2014
Date of Acceptance23-Apr-2015
Date of Web Publication16-Oct-2015

Correspondence Address:
X Zhou
Department of Microbiology, Nanchang Center for Disease Control and Prevention, Nanchang
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.167328

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 ~ Abstract 

Purpose: A diarrhoea outbreak occurred in a kindergarten, which caused 21 relevant infected cases. Our object was to confirm the pathogens and their molecular characterisation. Materials and Methods: Faecal samples from 21 patients were collected on the 3rd day after their symptom onset, and a regular epidemiological investigation was conducted. Bacterial isolation was performed in accordance with standard laboratory protocol, serological and molecular characterisations were determined by serum agglutination test and real-time polymerase chain reaction (PCR) method, respectively. The pulsed field gel electrophoresis (PFGE) and 16S rRNAs were conducted to determine the homology. Results: Eleven enteroinvasive Escherichia coli (EIEC) O136:K78 strains were isolated. The serum agglutination test showed that all strains' serotypes were E. coli (EIEC) O136:K78. Real-time PCR showed that 10 (91%) strains carried the invasion plasmid antigen H gene (ipaH), carried by all four Shigella species and EIEC. The strain that didn't carry the ipaH gene had different biochemical reactions of L-lizyna and L-rhamnose with the other strains. The complete 16S rRNA sequences showed 98.4% identity between ipaH-negative isolate and the others, and the PFGE indicated that the ipaH-negative isolate was not homological with other isolates in this diarrhoea outbreak. Conclusions: The diarrhoea outbreak was caused by E. coli (EIEC) O136:K78.


Keywords: 16S rRNA, enteroinvasive Escherichia coli, invasion plasmid antigen H, O136:K78, pulsed field gel electrophoresis


How to cite this article:
Zhou X, Xia W, Tu J, Xue L, Ni X. Molecular characterisation of enteroinvasive Escherichia coli O136:K78 isolates from patients of a diarrhoea outbreak in China. Indian J Med Microbiol 2015;33:528-32

How to cite this URL:
Zhou X, Xia W, Tu J, Xue L, Ni X. Molecular characterisation of enteroinvasive Escherichia coli O136:K78 isolates from patients of a diarrhoea outbreak in China. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 7];33:528-32. Available from: http://www.ijmm.org/text.asp?2015/33/4/528/167328



 ~ Introduction Top


Enteroinvasive  Escherichia More Details coli (EIEC) are a group of E. coli that may cause dysentery-like epidemics in humans. Both EIEC and Shigella possess a large (≈140 MDa) plasmid encoding several outer membrane proteins involved in invasion of epithelial cells.[1] The EIEC group is represented by isolates restricted to certain serogroups. Only a few serogroups have been found to be responsible for outbreaks of diarrhoea that is, O28ac, O112ac, O124, O136, O143, O144, O152, O164 and O167.[2] The EIEC corresponds to bioserotypes found in a dozen of E. coli serogroups.[3] Interestingly, some of these O antigens are identical or similar to Shigella O antigens.[4] It was known that the invasion plasmid antigen H gene (ipaH) is carried by all four Shigella species and EIEC, and its detection was usually used for diagnosis of dysentery caused by Shigella spp. and EIEC.[5],[6] Venkatesan et al. found ipaH gene was relatively more sensitive in detecting virulent as well as avirulent Shigella and EIEC compared with ipaB, ipaC, and ipaD genes.[7]

The role played by EIEC in endemic diarrheal disease has not been investigated extensively. However, some studies indicated that these bacteria can be isolated with relatively high frequency depending on the population investigated.[8] Toledo and Trabulsi conducted a study on the frequency of EIEC in children with diarrhoea in Sao Paulo, EIEC has been found in 5–7% of children living in medium-income families and in 20% of children who live in a slum, in the outskirts of the city.[3] A similar frequency has been reported by Echeverria et al. in Bangkok.[9] Outbreaks of foodborne infections due to EIEC have also been reported elsewhere.[10] There was a diarrhoea outbreak in a kindergarten in Nanchang, and 11 E. coli strains O136:K78 from 21 patients were isolated, this is firstly reported in this region. A series of experiments have been conducted to identify the pathogens that caused this diarrhoea outbreak, and their homology was studied with pulsed field gel electrophoresis (PFGE) and phylogenetic analysis of 16S rRNA sequences.


 ~ Materials and Methods Top


Bacterial isolates

A total of 11 EIEC strains were isolated from patients of an aggregating diarrhoea outbreak in a kindergarten in Nanchang, China in 2011. The bacterial strains used in this study were obtained from clinical specimens and were cultured by standard methods.

Biochemical test

The strains were grown on nutrient agar plate at 37°C overnight, and the suspensions were prepared with a turbidity equivalent to 0.5 McFarland. The suspensions were immediately inoculated into the ID 32E strips (bioMérieux, France) for 24 h. Biochemical profiles were obtained from ATB Expression identification software (Ref. 40 011 Biomerieux, France).

Preparation of chromosomal DNA

To isolate chromosomal DNA, one or two freshly grown colonies of bacteria were scraped into a 1.5 ml Eppendorf tube and resuspended in 500 μl of sterile water. The bacterial suspension was then boiled (at 100°C for 10 min) to release the DNA.

Real-time polymerase chain reaction to detect invasion plasmid antigen H gene

Invasion plasmid antigen H gene was detected by real-time polymerase chain reaction (PCR) in ABI 7300 using Shigella (ipaH gene) Fluorescent Polymerase Chain Reaction Diagnostic Kit (DAAN Gene Co., Ltd. of Sun Yat-sen University).

Pulsed field gel electrophoresis

Pulsed field gel electrophoresis was conducted according to protocols for the subtyping of E. coli O157:H7,  Salmonella More Details, and Shigella.[11]

Polymerase chain reaction amplification and sequencing of 16S rRNA

16S rRNA is amplified by PCR in a thermocycler (BIO-RAD PTC-200, USA). PCR was performed in a total volume of 50 μl, which contains 5 μl ×10 Taq reaction buffer, 1 μl dNTP Mix (10 mM of each dNTP), 1 μl of 10 μM each primer (F: 5'-AACACATGCAAGTCGAACG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'), 0.25 μl Taq DNA polymerase (Takara), and 10 pM bacterial DNA, then add Rnase-free water to 50 μl. The PCR products were examined by electrophoresis with a 1.0% agarose gel. Conditions for PCR amplification were: An initial denaturation step of 98°C for 5 min before 35 cycles of 95°C for 35 s, 55°C for 35 s, 72°C for 1.5 min, and then a final step of 72°C for 8 min for the last cycle. The PCR products were then purified, cloned and sequenced.

Phylogenetic analysis

The phylogenetic data described below were obtained by alignment and phylogenetic analysis of the bacterial sequences. The nucleotide sequences of 16S rRNA were aligned using the CLUSTAL W computer program.[12] A neighbour-joining analysis was used to reconstruct phylogenetic trees with the MEGA 4 software (Center for Evolutionary Medicine and Informatics, The Biodesign Institute, USA).[13]


 ~ Results Top


Biochemical profiles and serotpyes

All 11 strains were examined for chemical reaction in ID 32E strips. The results indicated that all 11 isolates were identified as E. coli, the biochemical profiles of strain 2012K10 and the other strains were 44465543400 and 04465543400, respectively. There were two different biochemical reactions of L-lizyna and L-rhamnose in strain 2012K10 as shown in [Table 1]. All 11 strains belong to E. coli O136:K78 according to the agglutination test.
Table 1: Chemical profile of 11 strains from people with diarrhoea

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Invasion plasmid antigen H gene

Invasion plasmid antigen H was detected in 10 of 11 (91%) strains isolated from people with diarrhoea. As the result of real-time PCR indicated, strain 2012K10 didn't carry ipaH gene [Figure 1].
Figure 1: Profile of invasion plasmid antigen H gene detection by real-time polymerase chain reaction in ABI 7300

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Pulsed field gel electrophoresis

Macrorestriction ploymorphism of genomic DNA determined by PFGE revealed the presence of two pulsotypes among the eleven study strains. Ten belonged to one and one (strain 2012K10) the other [Figure 2], which indirectly confirmed that the lack of ipaH gene in strain 2012K10. The result of PFGE indicated that the ipaH-negative isolate was not homological with other isolates in this diarrhoea outbreak.
Figure 2: Pulsed field gel electrophoresis profile of 11 E. coli strains from people with diarrhoea (Lane 2–3, 6–9, 12–14 and 16, strain 2012K1-2012K11; Lane 17, Shigella sp.; M, molecular weight marker)

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Phylogenetic analysis of 16s rRNA

Two representative strain 2010K10 (JQ951604.1) and 2010K11 (JQ951605.1) were subjected to PCR amplication of the 16S rRNA. The ampli.ed 16S rRNA gene, a region of 146 bp [Figure 3] , was cloned into the pUCm-T vector (Sangon, Shanghai, China), and subsequently subjected to bi-directional DNA sequencing (Sangon, Shanghai, China). Data for the phylogenetic analysis were obtained from sequences contained in the GenBank: EHEC Strain ATCC43895 (Z83205.1), Shigella sonnei strain AU65 (EF032687.1), E. coli strain SCDC-1 (HM576813.1), Shigella boydii strain 3052–94 (AY696681.1), E. coli O111: H-(GU237022.1), E. coli strain: JCM 24009 (AB548582.1) and E. coli strain LW1655F+ (AY616658.1). Alignment of the 16S rRNA nucleotide sequence was performed by the computer program MEGA 4.0. [Figure 4] shows the phylogenetic tree for these strains on the basis of their 16S rRNAs. The result indicates that strain 2010K10 is more closely related to S. sonnei than to E. coli, while strain 2010K11 is more closely related to E. coli O136:H-. Strain 2010K10 and strain 2010K11 have 98.4% similarity to each other, nucleotide sequence comparison of 16S rRNA between strain 2012K10 and 2012K11 is shown in [Figure 5].
Figure 3: Profile of 16s rRNA products in 3% agarose gel electrophoresis (Lane 1 and 2: Strain 2012K10 and 2012K11; N: Negative control; M: Marker)

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Figure 4: Phylogenetic tree based on the nucleotide sequences of 16S rRNA genes and the tree was constructed by the neighbour-joining method, using the MEGA 4.0 software. The scale indicates the percentage of base difference (percent divergence)

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Figure 5: Nucleotide sequence comparison of 16S rRNA gene between strain 2012K10 and 2012K11, the nucleotides that are not identical are highlighted

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 ~ Discussion Top


E. coli serotype O136:K78 was firstly described by Sakasaki and Namioka in Japan, 1957.[14] Fernandes and Trabulst identified an Escherichia isolate 193T-64 as E. coli O136:K78 by agglutination tests in 1969.[15] Nevertheless, E. coli O136:K78 was not extensively reported. In this study, 11 E. coli O136:K78 strains were isolated from patients with diarrhoea that occurred in a kindergarten. A series of methods were conducted to identify these strains, including biochemical profile and agglutinating tests. Profiles of PFGE indicated that strain 2012K10 was not homological with other strains in this diarrhoea outbreak. And it was interesting that strain 2012K10 did not carry ipaH gene, carried by all four Shigella species and EIEC, which was not reported before. Moreover, the biochemical profiles between strain 2012K10 and others were also slightly different.

Phylogenetic-tree analysis is often used as a method to classify organisms. In general, 16S rRNA is most frequently used for such analyses.[16] We found that Strain 2010K10 and strain 2010K11 have 98.4% similarity to each other, and they were closely related to S. sonnei and to E. coli, respectively. EIEC and Shigella have a very close relationship not only in the type of disease that these mircoorganisms cause, but also in cross-reaction between their O-antigens.[17] And we did find the cross-reaction between E. coli O136:K78 and Shigella sp. during the agglutination tests.

In summary, the strains that caused this diarrhoea outbreak have been identified by a number of methods. We found a very interesting difference of ipaH gene between strain 2012K10 and the others, which was not reported before as we know. Analysis of 16S rRNA sequences and PFGE supported that strain 2012K10 was not homological with other isolates in this diarrhoea outbreak. Nevertheless, the unsatisfactory part in our study is that we failed to collect food the patients shared in the kindergarten according to the epidemiology report, and all the specimens were only collected from patients in The Children's Hospital of Jiangxi Province. All in all, health and hygiene education need to be improved to lower the infectious risk among children in kindergartens in the future.


 ~ Acknowledgments Top


We sincerely appreciated the help that The Children's Hospital of Jiangxi Province gave in the process of collecting clinical specimen.

 
 ~ References Top

1.
Hale TL, Sansonetti PJ, Schad PA, Austin S, Formal SB. Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli. Infect Immun 1983;40:340-50.  Back to cited text no. 1
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2.
Orskov F, Orskov I. Serotyping of Escherichia coli. Methods Microbiol 1984;14:43-112.  Back to cited text no. 2
    
3.
Toledo MR, Trabulsi LR. Frequency of enteroinvasive Escherichia coli in children with diarrhea and healthy controls in Sao Paulo, Brazil. Rev Microbiol Sao Paulo 1990;21:1-4.  Back to cited text no. 3
    
4.
Ewing WH. Edwards and Ewing's Identification of Enterobacteriaceae. 4th ed. New York: Elsevier Science; 1986.  Back to cited text no. 4
    
5.
Vu DT, Sethabutr O, Von Seidlein L, Tran VT, Do GC, Bui TC, et al. Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam. J Clin Microbiol 2004;42:2031-5.  Back to cited text no. 5
    
6.
Sethabutr O, Venkatesan M, Murphy GS, Eampokalap B, Hoge CW, Echeverria P. Detection of Shigellae and enteroinvasive Escherichia coli by amplification of the invasion plasmid antigen H DNA sequence in patients with dysentery. J Infect Dis 1993;167:458-61.  Back to cited text no. 6
    
7.
Venkatesan MM, Buysse JM, Kopecko DJ. Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli. J Clin Microbiol 1989;27:2687-91.  Back to cited text no. 7
    
8.
Martinez MB, Whittan TS, McGraw EA, Rodrigues J, Trabulsi LR. Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups. FEMS Microbiol Lett 1999;172:145-51.  Back to cited text no. 8
    
9.
Echeverria P, Sethabutr O, Serichantalergs O, Lexomboon U, Tamura K. Shigella and enteroinvasive Escherichia coli infections in households of children with dysentery in Bangkok. J Infect Dis 1992;165:144-7.  Back to cited text no. 9
    
10.
Gangarosa EJ. Epidemiology of Escherichia coli in the United States. J Infect Dis 1978;137:634-8.  Back to cited text no. 10
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11.
Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 2006;3:59-67.  Back to cited text no. 11
    
12.
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673-80.  Back to cited text no. 12
    
13.
Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-9.  Back to cited text no. 13
    
14.
Sakazaki R, Namioka S. Studies on a new Escherichia coli type: O 136: K78 (B22). Jpn J Exp Med 1957;27:411-6.  Back to cited text no. 14
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15.
Fernandes MR, Trabulsi LR. Antigenic identity of culture 193T-64 and E. coli 0136:K78(B22). Rev Inst Med Trop Sao Paulo 1969;11:101-3.  Back to cited text no. 15
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16.
Blanco M, Blanco JE, Mora A, Rey J, Alonso JM, Hermoso M, et al. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain. J Clin Microbiol 2003;41:1351-6.  Back to cited text no. 16
    
17.
Staaf M, Weintraub A, Widmalm G. Structure determination of the O-antigenic polysaccharide from the enteroinvasive Escherichia coli O136. Eur J Biochem 1999;263:656-61.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]



 

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