A real-time polymerase chain reaction (PCR) assay, amplifying the genes encoding lactose permease (lacY) and invasion plasmid antigen H (ipaH), was run on 121 isolates phenotypically classified as Shigella spp., enteroinvasive Escherichia coli (EIEC), or EIEC O nontypable (ONT). The results were compared with data from a generic E. coli multiple-locus variable-number of tandem repeat analysis (MLVA) and a Shigella MLVA.
The real-time PCR verified all Shigella spp. (n = 53) as Shigella (lacY negative) and all EIEC O121 (n = 15) and EIEC O124 (n = 2) as EIEC (lacY positive). However, the real-time PCR typed EIEC O164 as either EIEC (n = 2) or Shigella (n = 2) and, thus, was not suited for classifying this group of isolates. Interestingly, the majority (42/47, 89.4%) of the EIEC ONT were classified as Shigella (lacY negative) by the real-time PCR, and in nearly all cases, (92.9%, 39/42) data from both MLVA assays supported these findings. Overall, in 94.7% (114/121) of the isolates, the results from the real-time PCR were substantiated by the results from the MLVA assays.
In conclusion, the real-time PCR assay was fast and accurate in differentiating Shigella spp. from EIEC, with the exception of the EIEC O164 group. This molecular assay was particularly pragmatic for the challenging EIEC ONT group.
Hale TL : Genetic basis of virulence in Shigella species. Microbiol Rev 55, 206–224 (1991)
Lima IF , Havt A, Lima AA: Update on molecular epidemiology of Shigella infection. Curr Opin Gastroenterol 31, 30–37 (2015)
Nyholm O , Lienemann T, Halkilahti J, Mero S, Rimhanen-Finne R, Lehtinen V, Salmenlinna S, Siitonen A: Characterization of Shigella sonnei isolate carrying Shiga toxin 2-producing gene. Emerg Infect Dis 21, 891–892 (2015)
Ojha SC , Yean Yean C, Ismail A, Singh KK: A pentaplex PCR assay for the detection and differentiation of Shigella species. Biomed Res Int 2013, 412370 (2013)
Kapperud G , Rorvik LM, Hasseltvedt V, Hoiby EA, Iversen BG, Staveland K, Johnsen G, Leitao J, Herikstad H, Andersson Y, et al.: Outbreak of Shigella sonnei infection traced to imported iceberg lettuce. J Clin Microbiol 33, 609–614 (1995)
Guzman-Herrador B , Vold L, Comelli H, MacDonald E, Heier BT, Wester AL, Stavnes TL, Jensvoll L, Lindegard Aanstad A, Severinsen G, Aasgaard Grini J, Werner Johansen O, Cudjoe K, Nygard K: Outbreak of Shigella sonnei infection in Norway linked to consumption of fresh basil, October 2011. Euro Surveill 16 (2011)
Heier BT , Nygard K, Kapperud G, Lindstedt BA, Johannessen GS, Blekkan H: Shigella sonnei infections in Norway associated with sugar peas, May–June 2009. Euro Surveill 14, (2009)
Rawal M , Hoff E, Aas-Pedersen L, Haugum K, Lindstedt BA: Rapid multiple-locus variable-number tandem-repeats analysis of Shigella spp. using multicolour capillary electrophoresis. J Microbiol Methods 83, 279–285 (2010)
Levine MM : Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis 155, 377–389 (1987)
Yang F , Yang J, Zhang X, Chen L, Jiang Y, Yan Y, Tang X, Wang J, Xiong Z, Dong J, Xue Y, Zhu Y, Xu X, Sun L, Chen S, Nie H, Peng J, Xu J, Wang Y, Yuan Z, Wen Y, Yao Z, Shen Y, Qiang B, Hou Y, Yu J, Jin Q: Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery. Nucleic Acids Res 33, 6445–6458 (2005)
Lan R , Alles MC, Donohoe K, Martinez MB, Reeves PR: Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp. Infect Immun 72, 5080–5088 (2004)
Fukiya S , Mizoguchi H, Tobe T, Mori H: Extensive genomic diversity in pathogenic Escherichia coli and Shigella Strains revealed by comparative genomic hybridization microarray. J Bacteriol 186, 3911–3921 (2004)
van den Beld MJ , Reubsaet FA: Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur, J Clin Microbiol Infect Dis 31, 899–904 (2012)
Ud-Din A , Wahid S: Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC) and their differentiation. Braz, J Microbiol 45, 1131–1138 (2014)
Ito H , Kido N, Arakawa Y, Ohta M, Sugiyama T, Kato N: Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp. Appl Environ Microbiol 57, 2912–2917 (1991)
Hsu BM , Wu SF, Huang SW, Tseng YJ, Ji DD, Chen JS, Shih FC: Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test. Water Res 44, 949–955 (2010)
Kingombe CI , Cerqueira-Campos ML, Farber JM: Molecular strategies for the detection, identification, and differentiation between enteroinvasive Escherichia coli and Shigella spp. J Food Prot 68, 239–245 (2005)
Zhao J , Kang L, Hu R, Gao S, Xin W, Chen W, Wang J: Rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens. Foodborne Pathog Dis 10, 896–903 (2013)
Pavlovic M , Luze A, Konrad R, Berger A, Sing A, Busch U, Huber I: Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp. J Appl Microbiol 110, 1245–1251 (2011)
Jorgensen HJ , Pfaller MA, Carroll KC, Funke G, Landry ML, Richter S, Warnock DW (2015): Manual of Clinical Microbiology, vol. 1, 11th Edition, ASM Press, Washington.
Brandal LT , Wester AL, Lange H, Lobersli I, Lindstedt BA, Vold L, Kapperud G: Shiga toxin-producing Escherichia coli infections in Norway, 1992–2012: characterization of isolates and identification of risk factors for haemolytic uremic syndrome. BMC Infect Dis 15, 324 (2015)
Barletta F , Mercado EH, Lluque A, Ruiz J, Cleary TG, Ochoa TJ: Multiplex real-time PCR for detection of Campylobacter, Salmonella, and Shigella. J Clin Microbiol 51, 2822–2829 (2013)
Pfaffl MW : A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45 (2001)
Lobersli I , Haugum K, Lindstedt BA: Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci. J Microbiol Methods 88, 134–139 (2012)
Sahl JW , Morris CR, Emberger J, Fraser CM, Ochieng JB, Juma J, Fields B, Breiman RF, Gilmour M, Nataro JP, Rasko DA: Defining the phylogenomics of Shigella species: a pathway to diagnostics. J Clin Microbiol 53, 951–960 (2015)
Johnson JR : Shigella and Escherichia coli at the crossroads: machiavellian masqueraders or taxonomic treachery? J Med Microbiol 49, 583–585 (2000)
Horakova K , Mlejnkova H, Mlejnek P: Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase. J Appl Microbiol 105, 970–976 (2008)
Denamur E , Picard B, Tenaillon O (2010). In: Bacterial Population Genetics in Infectious Disease, eds. Robinson DA, Feil EJ, Falush D, Wiley–Blackwell, Hoboken, pp. 269–286
Leonard SR , Lacher DW, Lampel KA: Draft Genome sequences of the enteroinvasive Escherichia coli strains M4163 and 4608-58. Genome Announc 3, (2015)
Kutyavin IV , Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J: 3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28, 655–661 (2000)
Rezwan F , Lan R, Reeves PR: Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences. J Bacteriol 186, 7460–7465 (2004)
Cheasty T , Rowe B: Antigenic relationships between the enteroinvasive Escherichia coli O antigens O28ac, O112ac, O124, O136, O143, O144, O152, and O164 and Shigella O antigens. J Clin Microbiol 17, 681–684 (1983)
Linnerborg M , Weintraub A, Widmalm G: Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross-reacting with Shigella dysenteriae type 3. Eur, J Biochem 266, 460–466 (1999)