1. 2. Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA
Molecular Epidemiology of Shiga toxin-producing Escherichia coli in the country of Georgia
Tea Tevdoradze1, E. Khmaladze1, G. Chanturia1, M. Lashkarashvili1, L. Tevzadze1 ,
N. Strockbine2
1. National Center for Disease Control and Public Health (NCDC), Tbilisi, Georgia.
2. Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) is a major cause of outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. STEC-associated HUS was first recognized in Georgia in 2009 following the diagnosis of a cluster of eight cases of HUS that resulted in 7 deaths in Tbilisi, Georgia. The study of these cases and also further research revealed the etiological role of non O157 STEC strains in the development of HUS in Georgia.
METHODS
DNA was extracted from both, broth and plated samples and tested on PCR analysis on existed molecular markers for STEC (stx1, stx2, eae, ehxA). For the isolates obtained in this study slide agglutination with the O104 single antisera (SSI diagnostics) and conventional Multiplex PCR assay for final confirmation and detection of O104 markers (stx2, terD, rfbO104, fliC H4) were performed. For identification of virulence factors of enteroaggregative E. coli EAEC multiplex assay (pCVD, AGGR) was applied.
For molecular genotyping of isolated strains Pulsed Field Gel Electrophoresis (PFGE) was applied. XbaI restriction endonuclease was used according to the PN standard protocol for non - O157 STEC.
Furthermore, whole genome sequencing of three STEC strains was performed by the next-generation sequencing (NGS) Illumina MiSeq platform. Obtained raw reads were further analyzed by the CLC Genomics Workbench (CLC Bio) software package. Whole-genome SNP phylogenetic tree was constructed with the EDGE bioinformatics software.
Sequence types were identified and the virulence and the resistance genes profiles were determined based on the database of Center for Genomic Epidemiology, Denmark (
RESULTS
Figure 1. Phylogenetic relationships among strains analyzed in this study.
In this study 102 diarrhea cases were seen and 28 were laboratory confirmed as STEC infection. 41 % of confirmed cases were complicated with HUS. Majority (17/28) of cases were stx2 positives or stx2 positives in combination with eae or ehxA genes.
Totally 13 STEC strains were isolated during this research period; eight STEC strains were genetically characterized. Five stx2 producing strains were identified which was confirmed as enteroaggregative O104 E. coli strains, while three strains were non O104 with presence of stx2/ ehxA.
SNP phylogenetic tree revealed that 0104:H4 strains were clustered in the subgroup with historical STEC isolates from Georgia (2009) and German strain from outbreak (2011). The third non-O104 STEC strain was identified as E. coli O174:H210174:H21 isolate and showed more genetic similarity with E. coli O152:H28 strain isolated in Japan in 2008.
Table 1. Clinical and Laboratory Findings for STEC Among Individuals with Acute Diarrhea in Tbilisi, Georgia from January, until September, 2015
Quarter 1 No of Cases (%)
Quarter 2 No of Cases (%)
Quarter 3 No of Cases (%)
Quarter 4 No of Cases (%)
Quarter 5
No of Cases (%)
Quarter 6
Quarters 1-6
Number of patients tested for STEC 12 17 38 4 5 26
102
Patients hospitalized 7
(58%)
(31%) 36
(95%) 3
(75%)
(80%) 24
(92%) 79
(77%)
Patients with HUS 2
(17%)
(12%) 9
(24%)
(60%)
(19%)
Patients positive for stx2
(0%) 8
(21%)
(50%)
(40%)
(16%) 18
(18%)
HUS patients positive for stx2
(56%)
(67%) 11
(46%)
Patients with any evidence of STEC: stx1/2, eae or ehxA
(29%)
(35%) 28
(27%)
STEC isolated 1 6
STEC/EAEC O104:H4 isolated
Table 2. Characteristics of the STEC strains based on O104 and enteroaggregative conventional multiplex PCR assays and multilocus sequence typing (MLST).
DISCUSSION
Isolate
Year
PCR Result
Allele no.
ST complex (ST)
adk
fumC
gyrB
icd
mdh
purA
recA
STEC Strain A
2012
O104 EAEC + 6 5
136 9 7
ST678 (678)
STEC Strain B
2015
STEC Strain C
2014
O104 EAEC - 95 15 18 8 14
ST677 (677)
In this study we tried to learn more on molecular characteristics of Shiga toxin-producing E. coli strains in Georgia. It should be noted that stx2-producing E. coli is the major cause of bloody diarrhea disease complicated with HUS in Georgia. In most of the cases the source of infection is enteroaggregative O104 which emphasizes the concern that this bacterium is widespread in the country. In addition, genetic diversity that was seen among STEC strains contributes to the growth of interest of the problem; based on study results we suggest that it is essential to perform additional in depth research.
Acknowledgements: The research study described in this presentation was conducted in the frame of the International Science and Technology Center (ISTC) project #G-2099, funded by the Biotechnology Engagement Program (BTEP) of US Department of Health and Human Services and International Science.