Antimicrobial Susceptibility Study
Molecular analysis and antimicrobial susceptibility of enterotoxigenic Escherichia coli from diarrheal patients

https://doi.org/10.1016/j.diagmicrobio.2014.10.008Get rights and content

Highlights

  • Several predominant enterotoxigenic Escherichia coli clones in Shanghai, China, were identified by both pulsed-field gel electrophoresis and multilocus sequence typing analysis in this study.

  • Isolates of different clones carried different plasmid and chromosome-encoded virulence factors.

  • Several multidrug-resistant clones were identified.

  • We identified a clone involved in nosocomial neonatal diarrhea, and it was also a multidrug-resistant clone.

Abstract

A total of 123 enterotoxigenic Escherichia coli (ETEC) isolates from diarrheal patients from June to December 2012 in Shanghai, China, were examined to determine their genetic relatedness using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) and for the presence of virulence genes and antimicrobial susceptibility. Twenty-nine sequence types (STs) and 63 PFGE patterns were identified, and results from the 2 subtyping methods correlated well. The 12 isolates of PFGE cluster B all belonged to ST-2332 and were associated with nosocomial neonatal diarrhea. Isolates of a cluster usually had the same set of virulence factors, whereas isolates of different PFGE clusters carried diverse combinations of virulence determinants. Isolates belonging to ST-2332 and ST-182 (n = 9) were resistant to at least 6 antimicrobials. Our findings highlighted the need of active surveillance programs for infectious diseases collecting data at both epidemiological and genetic levels that can detect high-risk lineages of pathogens in order to rapidly identify disease outbreaks.

Introduction

Enterotoxigenic Escherichia coli (ETEC) is frequently associated with travelers' diarrhea and a major cause of childhood diarrhea in developing countries (Qadri et al., 2005). A recent survey ranked ETEC (specifically heat-stable toxin–producing strains), as the third most important cause of moderate-to-severe diarrhea after rotavirus and Cryptosporidium in children aged 0–11 months in developing countries (Kotloff et al., 2013). ETEC accounts for about 9% of bacterial cases in foodborne disease outbreaks in the United States from 1998 to 2008 (Gould et al., 2013). Despite of the public health significance of ETEC, little is known about ETEC in China.

Human ETEC is able to adhere to and colonize the intestinal mucosa and cause profuse watery diarrhea and abdominal cramping. Diverse virulence factors have been identified to be involved in the ETEC pathogenicity. For example, 2 plasmid-encoded virulence factors, colonization factors (CFs) and enterotoxins (human heat-stable toxin, porcine heat-stable toxin, and/or heat-labile toxin [LT]) have been frequently associated with diseases. CFs promote adherence to and colonization of the host small intestine, whereas enterotoxins stimulate the lining of the intestines and induce watery diarrhea (Nataro and Kaper, 1998). At least 23 different CFs (CFA/I and CS1 to CS22) have been identified in human ETEC, among which CFA/I and CS1-6 are most prevalent (Gaastra and Svennerholm, 1996). ETEC produces one or both of LT and heat-stable toxin, which can, respectively, promote the cyclic levels of cyclic AMP and cyclic GMP (cGMP) through binding to intracellular adenyl-cyclase (Kaper et al., 2004, Nataro and Kaper, 1998). In addition, recent studies have looked into a number of chromosomal determinants that could be associated with ETEC virulence, such as ClyA (a hemolysin), LeoA (a cytoplasmic protein with GTPase activity that is required for LT secretion), EatA (a serine protease autotransporter), enteroaggregative heat-stable toxin 1 (EAST-1) (a toxin that share 50% identity with the enterotoxigenic domain of heat-stable toxin and can also trigger the cGMP signal transduction pathway), and Tia and TibA (2 outer membrane proteins that mediate adhesion and invasion) (Del Canto et al., 2011, Gonzales et al., 2013, Rivera et al., 2013).

Human ETEC can be genetically diverse. Molecular typing techniques allow the detection of diversity among isolates and the identification of clones that are epidemiologically significant. Isolates collected from different parts of the world have yielded over 42 different lineages (Steinsland et al., 2010) and 117 serotypes (Wolf, 1997). Pulsed-field gel electrophoresis (PFGE) is widely used for its great discriminatory power for lineage analyses (Al-Gallas et al., 2007, Regua-Mangia et al., 2004, Wu et al., 2007), whereas multilocus sequence typing (MLST) is increasingly being used for ETEC typing in recent years due to ease of data interpretation and comparison to other groups via a large database (Kjelstrup et al., 2013, Rodas et al., 2011a, Steinsland et al., 2010).

In this study, we conducted PFGE and MLST analysis in parallel to characterize genetic relatedness of ETEC from diarrhea patients in Shanghai, China, and determined their antibiotic susceptibilities and virulence factors in attempt to identify high-risk clones of ETEC.

Section snippets

Bacterial isolates

This study was conducted at 9 hospitals located in 5 districts of Shanghai from June to December 2012. Stool samples were collected from diarrhea patients (identified as at least 3 loose, liquid, or watery stools within 24 h) and inoculated onto SSI enteric medium (Statens Serum Institute, Copenhagen, Denmark) for detection of enteric pathogens. Five lactose-positive colonies were again picked from the medium and purified on MacConkey agar plates (SSI). Following overnight incubation, pink rose

MLST and PFGE analysis

From June to December 2012, a total of 3107 samples were collected from diarrhea patients, and 123 (4.0%) ETEC isolates were identified. Genetic relatedness of the 123 ETEC isolates was analyzed using both MLST and PFGE. MLST resulted in 29 STs (Fig. 1): ST-94 (n = 25) was the most prevalent, followed by ST-1491 (n = 13), ST-218 (n = 13), ST-2332 (n = 12), ST-182 (n = 9), ST-69 (n = 9), and ST-4 (n = 6). Five novel STs (ST-3930, ST-3931, ST-3932, ST-3933, and ST-3934) were identified. PFGE generated 63

Discussion

ETEC has been frequently reported as an important etiologic agent associated with diarrhea in developing countries. A retrospective study on human DEC in Zhejiang Province also highlighted the challenge of DEC infections in China (Chen et al., 2014). However, little is known about the genotypic and phenotypic characteristics of this pathogen in China.

Previous studies reported ETEC prevalence varies significantly by regions and across populations (Al-Gallas et al., 2007, Nada et al., 2011, Rodas

Acknowledgments

This work was funded in part by the National High Technology Research and Development Program of China (863 Program) (2012AA101601), the Mega-projects of Science and Technology Research of China (2012ZX10004215-003 and 2012ZX10004201), the Research Projects of Health and Family Planning Commission of Changning District of Shanghai (20134GW24001), and the Research Projects of Health and Family Planning Commission of Putuo District of Shanghai (PU-KW12314). We declare that we have no conflicts of

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