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Newly Emerged Serotype 1C of Shigella Flexneri

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Newly Emerged Serotype 1C of Shigella Flexneri G C A T T A C G G C A T genes Article Newly Emerged Serotype 1c of Shigella flexneri: Multiple Origins and Changing Drug Resistance Landscape Pawan Parajuli 1, Bui Quang Minh 2 and Naresh K. Verma 1,* 1 Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia; [email protected] 2 Research School of Computer Science & Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia; [email protected] * Correspondence: [email protected] Received: 28 July 2020; Accepted: 1 September 2020; Published: 3 September 2020 Abstract: Bacillary dysentery caused by Shigella flexneri is a major cause of under-five mortality in developing countries, where a novel S. flexneri serotype 1c has become very common since the 1980s. However, the origin and diversification of serotype 1c remain poorly understood. To understand the evolution of serotype 1c and their antimicrobial resistance, we sequenced and analyzed the whole-genome of 85 clinical isolates from the United Kingdom, Egypt, Bangladesh, Vietnam, and Japan belonging to serotype 1c and related serotypes of 1a, 1b and Y/Yv. We identified up to three distinct O-antigen modifying genes in S. flexneri 1c strains, which were acquired from three different bacteriophages. Our analysis shows that S. flexneri 1c strains have originated from serotype 1a and serotype 1b strains after the acquisition of bacteriophage-encoding gtrIc operon. The maximum-likelihood phylogenetic analysis using core genes suggests two distinct S. flexneri 1c lineages, one specific to Bangladesh, which originated from ancestral serotype 1a strains and the other from the United Kingdom, Egypt, and Vietnam originated from ancestral serotype 1b strains. We also identified 63 isolates containing multiple drug-resistant genes in them conferring resistance against streptomycin, sulfonamide, quinolone, trimethoprim, tetracycline, chloramphenicol, and beta-lactamase. Furthermore, antibiotic susceptibility assays showed 83 (97.6%) isolates as either complete or intermediate resistance to the WHO-recommended first- and second-line drugs. This changing drug resistance pattern demonstrates the urgent need for drug resistance surveillance and renewed treatment guidelines. Keywords: Shigella flexneri serotype 1c; bacteriophage; serotype evolution; multidrug resistance 1. Introduction Shigellosis or bacillary dysentery is caused by members of the Shigella genus, which are human-specific enteric pathogens. Shigella species are closely related to Escherichia coli but have specific virulence traits [1]. Historically, shigellosis was associated with significant morbidity and mortality during all major conflicts of the 19th and 20th centuries, during natural disasters, times of political upheaval, and subsequent refugee crises [2–4]. Shigellosis continues to be a major etiological agent of food and water-borne illness in many developing countries due to poor sanitation facilities and a lack of safe drinking water [5]. A recent study estimated that there are at least 190 million annual shigellosis cases and 70,000 deaths worldwide [6]. The majority of these deaths occurred in Sub-Saharan Africa and South Asia and involved children less than five years of age [7–9]. Recently, Shigella transmission dynamics have expanded to developed countries. This is largely due to increased international travel and sexual practices among men who have sex with men (MSM) [10]. Genes 2020, 11, 1042; doi:10.3390/genes11091042 www.mdpi.com/journal/genes GenesGenes2020 2020, 11, 11, 1042, x FOR PEER REVIEW 22 ofof 14 14 S. flexneri is the predominant species of Shigella causing shigellosis in developing countries in Shigella flexneri Shigella Asia [9], Latin Americais the[11,12] predominant and Africa [13]. species The S. of flexneri serotypes,causing except shigellosis for serotype in developing 6, consist countriesof a polysaccharide in Asia [9], backbone Latin America with the [11 repeating,12] and tetrasaccharide Africa [13]. The unitsS. flexneriof threeserotypes, rhamnoses except and an for N- serotypeacetylglucosamine 6, consist of (GlcNAc)a polysaccharide residue in backbone the O-antigen with component the repeating of the tetrasaccharide bacterial lipopolysaccharide units of three rhamnoses(LPS) [14]. and The an modificationsN-acetylglucosamine to the basic (GlcNAc) O-antigen residue backbone in the O-antigenby the addition component of glucosyl, of the bacterialO-acetyl, lipopolysaccharideor phosphoethanolamine (LPS) [groups14]. The to modifications one or more su togars the give basic rise O-antigen to at least backbone 19 serotypes by the of additionS. flexneri of[15] glucosyl, (Figure O-acetyl,1). or phosphoethanolamine groups to one or more sugars give rise to at least 19 serotypes of S. flexneri [15] (Figure1). Figure 1. Schematic representation of the lipopolysaccharide of Shigella flexneri serotype 1c. Each tetrasaccharideFigure 1. Schematicunit of O-antigen representation has one ofN -acetylglucosaminethe lipopolysaccharide (GlcNAc) of S. andflexneri three serotype rhamnose 1c. sugars Each (Rha).tetrasaccharide All serotype unit 1c of strains O-antigen have twohas glucosylone N-acetylglucosamine groups attached to (GlcNAc) GlcNAc and somethree rhamnose of them have sugars an III additional(Rha). Allacetyl serotype group 1c strains attached have to Rha two glucosyl. groups attached to GlcNAc and some of them have an additional acetyl group attached to RhaIII. S. flexneri serotype 1c is an emerging serotype in developing countries [16]. The serotype 1c was first identifiedS. flexneri during serotype the 1c 1980s is an and emerging since then, serotype it has in become developing the most countries common [16].S. The flexneri serotypeserotype 1c was in multiplefirst identified geographic during regions the 1980s [17 –and20]. since This then, serotype it has has become a unique the O-antigenmost common structure, S. flexneri comprised serotype of in α α amultiple disaccharide geographic(two-glucosyl regions group)- [17–20]. -D-GlcThis serotypep-(1→2)- has-D-Glc a uniquep linked O-antigen to O4 of structure, the GlcNAc comprised residue [ 21of]. a Thedisaccharide glucosylation (two-glucosyl in S. flexneri group)is performed - α-D-Glcp-(1 by➔ a2)- bacteriophage-acquiredα-D-Glcp linked to O4 of operon the GlcNAc containing residue three [21]. genes;The glucosylationgtrA, gtrB and in S.gtr(type) flexneri. is The performed products by of a bacteriophage-acquired first two genes, gtrA and operongtrB, containingare highly three conserved genes; amonggtrA, gtrB diff erentand gtr(S.type flexneri). Theserotypes products and of first are involvedtwo genes, in gtrA transferring and gtrB, the are glucosyl highly conserved group from among the cytoplasmdifferent S. to flexneri the periplasm serotypes [and22]. are The involvedgtr(type) in genetransferring (where the ‘type’ glucosyl refers group to S. from flexneri the serotype)cytoplasm is to uniquethe periplasm to each serotype[22]. The and gtr(type) encodes gene a serotype-specific (where ‘type’ refers glucosyltransferase to S. flexneri serotype) that attaches is unique the glucosyl to each groupserotype to a and specific encodes sugar a inserotype-specific the tetrasaccharide glucosyltr repeatansferase unit [23]. that The additionattaches the of two glucosyl glucosyl group groups to a isspecific due to sugar the presence in the tetrasaccharide of two separate repeatgtr operons unit [23]. in The the addition serotype of 1c two chromosome. glucosyl groups The additionis due to ofthe thepresence first glucosyl of two separate group is gtr mediated operons in by the the serotypegtrI operon 1c chromosome. carried bythe The SfI addition bacteriophage, of the firstwhich glucosyl is alsogroup the is basis mediated of O-antigen by the gtrI modification operon carried in S. by flexneri the SfIserotypes bacterioph 1aage, and which 1b. However, is also the the basis second of O- glucosylantigen modification group is mediated in S. flexneri by the serotypesgtrIc operon, 1a and carried 1b. However, by a diff erent,the second uncharacterized glucosyl group and isdistantly mediated relatedby the bacteriophagegtrIc operon, carried [24]. Recently, by a different, the complete uncharacteri genomezed analysisand distantly of S. flexnerirelated serotypebacteriophage 1c strain [24]. Y394Recently, showed the thatcomplete these genome two phage analysis regions of S. are flexneri arranged serotype as a cryptic1c strain prophage Y394 showed at 2 Mb that apart these [ 16two]. Furthermore,phage regions some are arranged of the serotype as a cryptic 1c strains prophage also haveat 2 Mb additional apart [16]. O-antigen Furthermore, modification, some of the due serotype to the addition1c strains of thealso O-acetyl have additional group mediated O-antigen by oacBmodificatgene,ion, encoded due to by the the addition bacteriophage of the Sf101O-acetyl [25]. group mediatedAlthough by oacB the serotypegene, encoded 1c is one by ofthe the bacteriophage most common Sf101S. flexneri [25]. serotypes in developing countries, the diversityAlthough and the evolution serotype of 1c serotype is one of 1c the remain most poorly common understood. S. flexneriUntil serotypes recently, in developingS. flexneri phylogeneticcountries, the studies diversity only and used evolution a handful of serotype of housekeeping 1c remain genespoorly [26 understood.,27]. Recently, Until sequencing recently, S. technologiesflexneri phylogenetic have provided studies only an unprecedented
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