Abstract
BackgroundAtypical Shigella flexneri Z variant, that agglutinate with E1037 group factor specific monoclonal antisera against Shigella flexneri IV-I but not with other group or type specific antisera, has continuously being isolated in Bangladesh since 1997. Later this serotype has been reported in Indonesia, China and Argentina. Despite being a provisional serotype, continuous isolation of these strains in diverse geographical regions implicated a great necessity to study the overall characteristics of these strains. Therefore, we extensively characterized S. flexneri Z strains using various phenotypic and molecular tools.MethodOf 3569 S. flexneri isolated between 1997 and 2015, 95 strains were identified as S. flexneri Z using a panel of polyvalent absorbed antisera and monoclonal antisera of S. flexneri (MASF). Of them, randomly selected 65 strains were molecular O-serotyped using multiplex PCR and characterized using different phenotypic and molecular techniques (i.e.biotyping, plasmid profile, virulence marker and PFGE) to determine relationship with other subserotypes of S. flexneri.ResultsAll these atypical S. flexneri Z strains were agglutinated with MASF B and IV-I antisera. Concordantly, these strains were positive to opt-gene, responsible for MASF IV-I sero-positive phenotype. However, molecular O-serotyping of all 65 strains could not differentiate between Z and Yb giving similar amplification products (wzx1-5 and opt). Contrarily, MASF based serotypic scheme distinguished among Z and Yb as well as Ya. All these S. flexneri Z showed typical biochemical reaction of S. flexneri, harboured a 140 MDa virulence plasmid and virulence markers namely ipaH, ial, sen, sigA and sepA genes. Along with the virulence plasmid, small plasmids (2.6, 1.8 and 1.6 MDa) were present as core plasmid. Moreover, a middle ranged plasmid and a 4.0 MDa sized plasmid were observed in 65% and 20% strains, respectively. Analysis of PFGE on XbaI-digested chromosomal DNA of Bangladeshi strains showed that S. flexneri Z had a close relatedness with Ya and Yb but completely different than the strains of Xa, Xb, 2a and 2b. This observation was found to be unequivocal while the overall result of biotyping, plasmid profile, and virulence factors was compared. Therefore, we conclude that these atypical serotype Z isolated in Bangladesh had a clonal relationship with Ya and Yb of Bangladesh and the opt gene played an important role in serotypic switching among them. Current serotyping scheme of S. flexneri strains fails to place many such atypical strains (1c, 1c+6, 1d, type 4, and 4c) including S. flexneri Z isolated from different parts of the world. Therefore, an updated serotyping scheme for identification of subserotypes of S. flexneri has been proposed to avoid multiple naming of the same subserotype having similar agglutination pattern.
Highlights
Shigellosis is one of the major causes of morbidity and mortality in developing countries like Bangladesh, especially among children under five years [1,2,3]
Randomly selected 65 strains were molecular O-serotyped using multiplex polymerase chain reaction (PCR) and characterized using different phenotypic and molecular techniques (i.e.biotyping, plasmid profile, virulence marker and Pulsed-field gel electrophoresis (PFGE)) to determine relationship with other subserotypes of S. flexneri. All these atypical S. flexneri Z strains were agglutinated with monoclonal antisera of S. flexneri (MASF) B and IV-I antisera
Atypical strains or newer subserotypes of S. flexneri are being isolated from different parts of the world, including Bangladesh [9, 10, 18]
Summary
Shigellosis is one of the major causes of morbidity and mortality in developing countries like Bangladesh, especially among children under five years [1,2,3]. The Global Burden of Disease Study 2015 estimates that Shigellosis causes 164,410 deaths worldwide in which 98.5% occurred in low and middle income countries and 33% in children under 5 years old [4]. Atypical Shigella flexneri Z variant, that agglutinate with E1037 group factor specific monoclonal antisera against Shigella flexneri IV-I but not with other group or type specific antisera, has continuously being isolated in Bangladesh since 1997. Later this serotype has been reported in Indonesia, China and Argentina. We extensively characterized S. flexneri Z strains using various phenotypic and molecular tools
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