Characterization of a serologically atypical Shigella flexneri Z isolated from diarrheal patients in Bangladesh and a proposed serological scheme for Shigella flexneri

Background 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. 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. Method Of 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. Results All 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.


Introduction
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]. A shigellosis survey between 2000 and 2004 in six Asian countries including Bangladesh, China, Pakistan, Indonesia, Vietnam and Thailand showed that the overall annual incidence of treated shigellosis was 2.1/1000 per year in all ages and 13.2/1000 per year in children under 5 years old, which was much higher than that in industrialized countries [2]. 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]. The disease is caused by Shigella spp. that colonizes the intestinal mucosa and continues to threaten public health mainly in less developed countries with conditions of poor sanitation. Moreover, clinical infection can be carried out by as little as 10 Shigella organisms even without neutralization of gastric acid [5]. Despite a significant global burden of the disease and worldwide spread of antibiotics resistance, no effective vaccine against shigellosis is widely available except for a few candidate vaccines that are under development [6]. The genetic and epidemiological switching leading to chronologic variability between serotypes, and emergence of atypical and new variants strains throughout the world often complicates the development of an effective vaccine [7][8][9][10][11]. As immunity to Shigella is serotype specific, vaccine protection will therefore depend on the representation of each serotype in the vaccine or at least the predominant serotypes. Therefore, estimating the disease burden due to Shigella infection and understanding it's epidemiology in countries where the disease is mostly prevalent have utmost importance [12][13][14].
Based on biochemical and serological properties, the genus Shigella is divided into four species: S. dysenteriae, S. flexneri, S. sonnei, and S.boydii. S. flexneri causes far more cases of dysentery than other species of Shigella in developing countries including Bangladesh [9,15]. A set of absorbed rabbit antisera against the type specific (I-VI) antigenic factors and group specific (3.4, 6, 7.8) factors are routinely being used for serotyping of S. flexneri that subdivide isolates into 13 serotypes (1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, Y and 6) [16,17]. However, this serological scheme of S. flexneri is not comprehensive since this cannot type several atypical and novel subserotypes [10,18]. In addition, the absorbed antisera often show residual cross-reactivity or are of lower titer since there are vast structural similarities between different serotypes of S. flexneri [19]. To overcome this constrain, monoclonal antisera of S. flexneri (MASF) can be used instead of the commercially available absorbed antisera [20]. Until recent, six type specific (I, II, IV-2, V, VI and 1c) and four group specific (Y-5, 6, 7.8 and IV-I) monoclonal antibodies are being developed in order to type all the existing subserotypes of S. flexneri including nontypeable strains [16,17].
A Wzy/Wzx dependent pathway, common to all S. flexneri except for S. flexneri type 6, synthesize the common repeating tetrasaccharide unit (referred to serotype Y), to which type and/ or group specific determinants including a-D-glucopyranosyl and/or O-acetyl and/or phosphoethanol amine (PEtN) groups are added exhibiting variation in O antigen [21][22][23]. These modifications are mostly conferred by prophage-encoded glucosyltransferase and/or acetyltransferase genes [24][25][26]. Recently a plasmid (6.8 Kb) mediated opt gene conferring the synthesis of a group antigen E1037 has been reported [22], albeit the antigen was first identified in 1984 in a provisional strain E1037 of S. flexneri by Berhard Rows [27]. Later, the antigen was detected in some strains of serotypes 4a, X, Y and 6 using E1037 specific antiserum, MASF IV-I [20]. Immunogenetical studies showed that the optO gene encoding the phosphoethanol amine transferase enzyme that transfers ethanolamine phosphate group predominantly to Rha II and rarely to both Rha II and/or Rha III residues of O antigen [28]. Until recently eight serotype converting genes (gtrI, gtrII, gtrIV, gtrV, oac, gtrX, opt and gtrIc) responsible for the transfer of α-D-glucopyranosyl, O-acetyl and phosphoethanol amine (PEtN) have been identified [24,29]. Based on the presence of these genes, PCR based O-serotyping technique was developed for the rapid detection of serotypes of S. flexneri [29,30].
Isolation of uncommon serotypes and subserotypes of Shigella spp. particularly of S. flexneri is not a rare event. Atypical strains or novel subserotypes (i.e. S. flexneri 1d, type 4, 7a and 7b etc.) are being isolated in different parts of the world including Bangladesh. Even several atypical serotypes became predominant over the established serotypes in developing countries [9,10,18]. For example, in China, the predominant serotype of S. flexneri 2a was replaced by atypical strain of S. flexneri (-:7.8, E1037) in 2010 [31]. Despite their epidemiological importance, these serotypes are not included in the current serological scheme. Use of monoclonal antisera may provide an extended serological scheme covering these atypical strains which remain in provisional status [18,[32][33][34]. Among the provisional strains of S. flexneri, a group of atypical strains that agglutinate only with MASF IV-1 but with any type or group antigen-specific antisera tested have been reported earlier (previously reported as 4X) and have continuously being isolated in Bangladesh since 1997 [10]. Later, this serologically atypical strain (as 4X) has also been reported from Indonesia and Argentina in 2002 and in 2010, respectively. Recently, isolates with the same serological characteristics have been reported (provisionally designated as serotype 4s) form China [35][36][37]. We renamed this atypical strain as S. flexneri Z for the systemic nomenclature of Shigella flexneri. This study was aimed to characterize the Bangladeshi strains of S. flexneri Z using different phenotypic and molecular techniques. In addition, we proposed an updated MASF based serotyping scheme for the identification of all typical and atypical serotypes and sub-serotypes of S. flexneri.

Ethics statement
This study was exempted from institutional review board (IRB) approval at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) given that no experimental procedures were performed and no patient or subject identifiers were collected.

Bacterial strains
A total 3569 of S. flexneri strains were isolated and identified from patients with diarrhoea attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) following the standard microbiological and biochemical methods [38], between January 1997 and December 2015. In this study, randomly selected 65 strains of S. flexneri Z [-:E1037] variants were included for characterization using different phenotypic and genotypic techniques and compared the same with other subserotypes of S. flexneri obtained from our laboratory collection. All strains were grown in trypticase soy broth with 0.3% yeast extract (TSBY) and stored at -70˚C after adding 15% glycerol. YSH6000 S. flexneri 2a, having 140MDa invasive plasmid, were used as positive control in the polymerase chain reaction (PCR) for detection of ipaH gene, ial, Shigella enterotoxin gene (set) and Serine protease autotransporter of Enterobacteriaceae (SPATE) genes (sigA, pic, sat and sepA) while an E. coli (ATCC 25922) strain lacking 140 MDa plasmid serves as a negative controls in PCR and also antibiotic susceptibility testing.

Serotyping
Serotyping of these isolates was performed using (i) a commercially available antisera kit (Denka Seiken, Tokyo, Japan) specific for all type-and group-factor antigens and (ii) a panel of monoclonal antisera (Reagensia AB, Stockholm, Sweden) reagents specific for all S. flexneri type-and group-factor antigens [10]. For the serotypic designation, we followed an extended version of serotyping scheme described in Table 1. PCR based assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes: gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, gtrX, and opt were used to detect the molecular serotype of S. flexneri according to procedure described elsewhere [29,30]. Characterization of S. flexneri Z isolated from Bangladesh and a proposed serological scheme of S. flexneri

Biochemical characterization
The biochemical reactions of the strains were performed by standard biochemical methods [38].

Isolation of plasmid DNA
Plasmid DNA was prepared according to the alkaline lysis method of Kado and Liu with some modifications described elsewhere [33,40]. The molecular weight of the unknown plasmid DNA was assessed by comparing with the mobility of the known molecular weight plasmids [41].

Determination of the role of transferable plasmid factor
Three strains of S. flexneri Z, K-5851 (AMP R SXT R NAL S ), KD-1170 (AMP R SXT R NAL S CAZ R-CRO R AMC R ), and K-7468 (AMP R SXT R NAL S CAZ R CRO R AMC R ) were selected as donor strain for the conjugation experiment. Each of the selected donor strains were mated with a recipient strain E. coli K-12 (lac + NAL R F -) according to the method described previously [33]. Transconjugants colonies were selected on the MacConkey agar plates containing ampicilin (100 mg/L) and nalidixic acid (50 mg/L) and the transfer frequency of the resistance plasmid was calculated by a method described earlier [42]. Plasmid profile analysis, plasmid curing and antimicrobial susceptibility test of the transconjugants strains were performed to determine the transfer of plasmids with antibiotic resistance phenotype.

Pulsed-field gel electrophoresis (PFGE)
To determine the clonal relationship of S. flexneri Z variant and closely related subserotypes, 29 representative strains of S. flexneri Z were compared with 13 strains of S. flexneri Ya, three S. flexneri Yb, three S. flexneri Xa and one strains of Xb using PFGE typing. Intact agarose embedded chromosomal DNA was prepared according to the guideline of pulsenet [45,46]. Genomic DNA was digested with the XbaI restriction enzyme for 4 h at 37˚C and the restriction fragments were separated by using CHEF-MAPPER system apparatus (Bio-Rad Laboratories) under the following conditions: switching time from 5 s to 35 s at 6 V cm −1 for 18 h at 14˚C. PFGE images were analyzed using the fingerprint analysis software BioNumerics version 4.5 (Applied Maths; Kortrijk, Belgium).The dendrogram constructed using the PFGE patterns was generated by the UPGMA algorithm with the Dice-predicted similarity value of two patterns at 1.0% pattern optimization and 1.5% band position tolerance.

Serological typing
All the isolates of S. flexneri were confirmed using the MASF B antisera in slide agglutination reaction suggesting that these strains were subserotypes of S. flexneri. Among 3569 S. flexneri, 95 isolates (2.7%) were identified as the atypical strains of S. flexneri, designated as S. flexneri Z that strongly agglutinated only with MASF IV-I but did not agglutinate with other group or type specific antisera of commercially available both absorbed polyvalent and monoclonal antisera. These 95 strains of S. flexneri Z neither agglutinated with type specific antisera nor with group specific antisera available in commercial antisera kit. The isolation rate of S. flexneri Z was reduced from 9.7% in 1997-2000 to 1.1% in 2013-15 (Table 2).

Molecular serotyping
Of the 95 S. flexneri Z isolates, 65 were randomly selected for molecular serotyping using multiplex PCR. All the 65 isolates were positive for wzx 1-5 gene which is commonly found in all S. flexneri, except for serotype 6. Additionally, opt gene was detected in all the S. flexneri Z isolates. We detected both wzx 1-5 and opt genes in all strains of S. flexneri Z and Yb.  Characterization of S. flexneri Z isolated from Bangladesh and a proposed serological scheme of S. flexneri

Biochemical characterization
All S. flexneri serotype Z isolates possessed the biochemical characteristics typical of S. flexneri, including negative reactions in utilization of sodium acetate, rhamnose, xylose, raffinose, ornithine, arginine, and lysine [47]. Around 58% of the isolates showed positive reaction in arabinose fermentation while 70% showed positive reaction in trehalose fermentation at variable time intervals. Interestingly, all the S. flexneri Z, Ya and Yb isolates produced indole after utilizing tryptophan, which was in contrast to the strains of S. flexneri 2a, 2b, Xa and Xb. Based on the biochemical reaction patterns S. flexneri Z isolates appeared to be more similar to S. flexneri Ya and Yb (Table 3).

Antibiotic susceptibility test
Of the 65 S. flexneri Z isolates tested for antibiotic susceptibility, 61% were resistant to amplicillin, followed by 57% of SXT, 55% to nalidixic acid and 41% to ciprofloxacin. Around 45% of isolates were resistant to three or more classes of antibiotics and thus identified these as multi drug resistant (MDR). Two isolates (K-7468 and KD-1170) were resistant to third generation cephalosporin including ceftriaxone, ceftazidime and amoxicillin-clavulanic acid. Both isolates were positive for extended spectrum beta lactamase (ESBL) as detected by using double disc synergy method. None of the isolates were resistant to nalidixic acid and ciprofloxacin.

Plasmid profile analysis
Analysis of plasmid DNA revealed that all the strains of S. flexneri Z harbored the 140 MDa plasmid along with three or four small sized (<6 MDa) plasmids (Fig 1). Additionally, a middle ranged plasmid (20-

Determination of the role of transferable plasmid
Three strains of S. flexneri Z with different plasmid patterns were selected for conjugation experiment with E. coli strain K-12 (Lac + F -NAL R ). After conjugation, a plasmid of 36 MDa from both K-7468 and KD-1170 strains was transferred to the recipient strain of E. coli K12 with the full spectrum of resistance to AMP, SXT, CRO, CAZ and AMC. Transfer frequency for both isolates were similar. However, in case of K-5851, a plasmid of 62 MDa was transferred with a frequency of 10-fold higher (6×10− 4 ) than the previous two isolates with resistance to AMP and SXT only ( Table 5). Transfer of antimicrobial resistance through transmissible plasmids was confirmed by curing the plasmids of transconjugants.

Detection of virulence and SPATE genes by PCR
ipaH, ial, sen, sigA and sepA genes were detected in all the tested S. flexneri Ya, Yb and Z strains, but none of the isolates were found to be positive for shet1, sat, pic and pet genes. However, all these nine genes were detected in each of the strains of serotype Xa, Xb, 2a and 2b.

Pulsed-field gel electrophoresis
The dendrogram generated based on Xba1-digested PFGE banding patterns demonstrated that all the 29 strains of S. flexneri Z isolated from Bangladesh were formed a single cluster (cluster 1) at 80% similarity level which was further divided into four subclusters (1A, 1B, 1C and 1D at 85%). Strains in each of the subclusters were grouped and intermingled with strains of Ya and Yb serotypes. On the other hand, serotypes 2a, 2b, Xa and Xb grouped in cluster 2 were completely different from the cluster 1 (Fig 2).

Discussion
Diversity of O antigen in S. flexneri evolves due to presence of serotype converting genes on horizontal transmissible elements [24,29]. These genes encode enzymes which modify the Characterization of S. flexneri Z isolated from Bangladesh and a proposed serological scheme of S. flexneri common tetrasaccharide unit (except serotype 6) of S. flexneri. As a result, atypical strains or newer subserotypes of S. flexneri are being isolated from different parts of the world, including Bangladesh [9,10,18]. In 2001, we reported three atypical strains of S. flexneri namely type 4, 1c and 4X which could not be definitively serotyped using commercially available antisera [10]. Therefore, for the identification of subserotypes of S. flexneri, a set of MASF antisera were used for a definitive serotyping of S. flexneri that are not included in the current serotyping scheme of S. flexneri. A group of atypical strains, designated as S. flexneri Z (previously reported as 4X), that only agglutinated with MASF IV-I but not with any other type or group specific monoclonal antibodies, were isolated in Bangladesh [10]. These atypical strains were also reported from Indonesia and Argentina but not characterized in detail [2,36]. In 2010, this serotype (provisionally designated as 4s) was reported from China having clonal relationship with serotype 2, Xa, Xb, Ya and Yb [35,37]. Since characterization of the atypical serotype isolated in Bangladesh remained undone, in this study, a comparative study was carried out to determine their relationship with other serotype of S. flexneri using different typing tools based on phenotypic and molecular traits. S. flexneri Z isolates could not be serotyped using absorbed antisera that are commercially available (Denka Seiken co. Japan), but only agglutinated with MASF IV-I group factor (Table 1). The opt gene, responsible for MASF IV-I sero reactive phenotype was detected in all the S. flexneri Z tested. Although PCR based detection of O-serotype of S. flexneri and serotyping using monoclonal antisera had showed consistent result, but it could not differentiate between the Yb and Z serotypes that gave similar amplification result (wzx1-5, opt). On the other hand, antisera based serotyping could differentiate between these two serotype Yb and Z, since MASF Y-5 specifically agglutinated with Yb strains, but not with Z strains. Since no genetic determinants for Y-5 is available, molecular serotyping of these isolates was not possible. Another limitation of the PCR based serotyping is that due to presence of mutation in Oantigen genes, this method may give discrepant results when cross checked with antisera based serotyping [48]. Moreover, for establishing a novel serotypes, producing and verifying a new antisera for the phenotypic schemes are much easier than identifying and validating a new primer pairs of the atypical strain [48]. Therefore, antisera based serotyping scheme is by far the gold standard for serotyping of S. flexneri [48]. It has been reported that a plasmid (6.85-10Kb)-borne opt gene may transfer to other serotypes via conjugation and eventually can convert their hosts into a MASF IV-I reactive phenotype [37]. Interestingly, we found a similar sized plasmid (~6.4 Kb) in 25% (13/52) strains of S. flexneri Z. However, a large proportion (75%) of the isolates was negative for this plasmid indicating that other mechanism might be involved in the acquisition and/or transmission of the gene, which accentuates further genetic investigation. Acquisition and transfer of plasmids carrying antibiotic resistance occur quite frequently in Shigella spp We found a plasmid of 62 Characterization of S. flexneri Z isolated from Bangladesh and a proposed serological scheme of S. flexneri MDa in size conferring resistance to ampicilin and SXT with a high degree of transfer frequency (6×10 −4 ). Interestingly, two strains of S. flexneri Z harbored a 36 MDa transmissible plasmid carrying resistance to 3 rd generation cephalosporin (ceftriaxone and ceftazidime) along with AMP and SXT, though the transfer frequency was 10-fold lower than that of the 62 MDa plasmid. Emergence of multiple drug resistance genes and their dissemination among these bacteria complicates the treatment strategy. Hence, more emphasis should be given to new/atypical isolates of Shigella spp.
In addition to the transferrable plasmids, all strains of S. flexneri Z harboured a 140 MDa plasmid along with three small sized stable plasmids (2.6, 1.8 and 1.6 MDa). Although the invasive plasmid of S. flexneri (140MDa) is extensively studied, little is known about the function of the small plasmid which constitute as a stable DNA pool. Therefore, these stable plasmids can be used as a molecular typing tool for the characterization of S. flexneri [41]. Overall analysis of plasmid DNA indicated that plasmid patterns of S. flexneri serotype Z isolates were indistinguishable from that of the serotypes Ya and Yb but distinct from those of serotype X and serotype 2. This observation was also true for biochemical properties, distribution of virulence genes and PFGE banding patterns of the isolates belonged to respective subserotypes and serogroups (Fig 2). The presence of similar plasmid pattern in isolates of different serotypes which are biochemically and genetically related to each other suggests that plasmid profiles may be a useful tool for determining their clonal relatedness.
All strains of serotype Z had biochemical characteristics typical of S. flexneri and very similar to that of serotypes Ya and Yb. Unlike serotype 2a, 2b, Xa and Xb, all strains of Z as well as Ya and Yb produced indole from tryptophan (Table 3). Although little is known about the effect of tryptophan utilization and indole production on characteristics of Shigella spp. it can be used as a biomarker for detection of S. flexneri Z. The dendrogram based on PFGE banding pattern was a better fit with indole production properties in which all strains belonged to clus-ter1 (Ya,Yb and Z) produced indole whereas strains of cluster 2 (2a, 2b, Xa and Xb) did not. Furthermore, strains in cluster 1contained relatively less virulence factors (ipaH, ial, sen, sigA and sepA genes), compared to the strains in cluster 2 (ipaH, ial, set1, sen, sat, pic, sepA and sigA genes).
In summary, Bangladeshi isolates of S. flexneri Ya, Yb and Z were different from isolates of serotypes 2a, 2b, Xa and Xb both at phenotypic and genotypic levels (Fig 2). Although Chinese isolates of the serotype 4s (= serotype Z) have the similar sero-agglutination pattern like S flexneri Z, Chinese strains showed clonal relationship with serotype 2, Xa, Xb, Ya and Yb [35][36][37]. This characteristic differences between isolates from two different regions explained that Bangladeshi isolates of S. flexneri Z may be emerged from a different clonal population than that of the Chinese isolates. This may be attributed to the insertion of gene carrying MASFIV-1 seroproperty into serotype Ya of Bangladeshi isolates through horizontal gene transfer mechanism.
S. flexneri Z and a few other atypical S. flexneri have been reported from different parts of the world that require further serological classification [9,10,17,18,22,30,35]. In order to meet this growing demand, we proposed an updated serotyping scheme for the identification of subserotypes of S. flexneri. Using this MASF based serological scheme it is possible to identify all the reported serotypes and subserotypes of S. flexneri including the atypical ones (Table 1). PMCPMC2557719 1997 on Shigella infection. The purpose of the review is to provide data on the global morbidity and mortality caused by the infection and to plan strategies of prevention and treatment. The data obtained from this literature were used to calculate the number of Shigella infection cases and the associated mortality occurring worldwide each year, by age and by clinical category. The burden of Shigella infection was also estimated by serogroup and serotype. A sensitivity analysis was performed to estimate the high and the low range of morbid and fatal cases in each category (mild cases remaining at home, moderate cases requiring outpatient care and severe cases demanding hospitalization). The result of the calculations and analysis revealed that the annual number of Shigella infections throughout the world was estimated to be 164.7 million. 163.2 million occurred in developing countries, with 1.1 million deaths, and 1.5 million occurred in industrialized countries. More than half of the episodes and death affects children under 5 years of age. In comparing developing countries against industrialized countries, the median of isolates are S. flexneri (60% vs. 16%), S. sonnei (15% vs. 77%), S. dysenteriae (6% vs. 1%), and S. boydii (6% vs. 2% 4. Collaborators GMaCoD. Global, regional, and national life expectancy, all-cause mortality, and causespecific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of