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Introduction . Virulence factors (VFs) are the most potent weapon in the molecular armoury of Shigella . In bacteria, the mobile genetic elements (MGEs) are contributors to the evolution of different types of clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR- cas ) variants and plasmid incompatibility types. The present study explored the virulence potential of Shigella in relation to the CRISPR- cas pattern and incompatibility types among the isolates. Hypothesis/Gap Statement. The profile of the CRISPR- cas systems among clinical isolates of Shigella in India has not been reported earlier. Limited knowledge is available on the pattern of plasmid incompatibility groups among clinical isolates Shigella . The bias is always towards studying the genetic elements associated with AMR, but the present study highlights CRISPR- cas and incompatibility types among Shigella in association with virulence. Aim. We aimed to investigate the distribution of virulence factors, CRISPR- cas pattern followed by plasmid incompatibility types among Shigella isolates. Methodology. Between 2012-2017, a total of 187 isolates of Shigella were included in the study. The virulence genes' distribution was carried out. CRISPR- cas profiling followed by analysis of the repeats and spacers was carried out. PCR-based replicon typing was used to determine the incompatibility types. The interplay was statistically determined using STATA. Results. The distribution of virulence genes showed varied pattern with ipaH present in all the isolates followed by ompA (93.6 %), virF (66.8 %), ial and s en (60.4 %), set1A (39.6 %) and set1B (39 %). CRISPR 1, CRISPR 3 and Cas6-Cas5 region were dominantly conserved. Twenty-two types of spacers were identified. The CRISPR3 repeat appeared to have a highly conserved sequence. CRISPR2 being the least common CRISPR type showed a strong association with an array of virulence genes ( ial-set1A-set1B-virF ) while CRISPR1 being the most dominant showed the least association with virulence genes ( sen-virF ). The dominant plasmids were found to be belonging to the inc FII group. The incompatibility groups FII, IncIγ, U, FIIS, FIIK, K, A/C, I1alpha was found to be associated with a greater number of virulence genes. Conclusion. The isolates showed increasing diversity in their gene content that contributes to increasing heterogeneity among the isolates, which is a known virulence strategy among pathogens.

Shigella is the second leading cause of diarrheal deaths worldwide. Azithromycin (AZM) is a potential treatment option for Shigella infection; however, the recent emergence of AZM resistance in Shigella threatens the current treatment strategy. Therefore, we conducted a comprehensive whole genome-based approach to identify the mechanism(s) of AZM resistance in Shigella . We performed antimicrobial susceptibility tests, polymerase chain reaction (PCR), Sanger (amplicon) sequencing, and whole genome-based bioinformatics approaches to conduct the study. Fifty-seven (38%) of the Shigella isolates examined were AZM resistant; Shigella sonnei exhibited the highest rate of resistance against AZM (80%). PCR amplification for 15 macrolide resistance genes (MRGs) followed by whole-genome analysis of 13 representative Shigella isolates identified two AZM-modifying genes, mph (A) (in all Shigella isolates resistant to AZM) and mph (E) (in 2 AZM-resistant Shigella isolates), as well as one 23S rRNA-methylating gene, erm (B) (41% of AZM-resistant Shigella isolates) and one efflux pump mediator gene, msr (E) [in the same two Shigella isolates that harbored the mph (E) gene]. This is the first report of msr (E) and mph (E) genes in Shigella . Moreover, we found that an IncFII-type plasmid predominates and can possess all four MRGs. We also detected two plasmid-borne resistance gene clusters: IS 26 - mph (A)- mrx (A)- mph (R)(A)-IS 6100 , which is linked to global dissemination of MRGs, and mph (E)- msr (E)-IS 482 -IS 6 , which is reported for the first time in Shigella . In conclusion, this study demonstrates that MRGs in association with pathogenic IS 6 family insertion sequences generate resistance gene clusters that propagate through horizontal gene transfer (HGT) in Shigella . IMPORTANCE Shigella can frequently transform into a superbug due to uncontrolled and rogue administration of antibiotics and the emergence of HGT of antimicrobial resistance factors. The advent of AZM resistance in Shigella has become a serious concern in the treatment of shigellosis. However, there is an obvious scarcity of clinical data and research on genetic mechanisms that induce AZM resistance in Shigella , particularly in low- and middle-income countries. Therefore, this study is an approach to raise the alarm for the next lifeline. We show that two key MRGs [ mph (A) and erm (B)] and the newly identified MRGs [ mph (E) and msr (E)], with their origination in plasmid-borne pathogenic islands, are fundamental mechanisms of AZM resistance in Shigella in Bangladesh. Overall, this study predicts an abrupt decrease in the effectiveness of AZM against Shigella in the very near future and suggests prompt focus on seeking a more effective treatment alternative to AZM for shigellosis.

Enteropathogenic Escherichia coli (EPEC) and Shigella are etiologic agents of diarrhea in children <5 years old living in resource-poor countries. Repeated bouts of infection lead to lifelong morbidity and even death. The goal of this study was to characterize local mucosal immune responses in Shigella - and EPEC-infected children <5 years of age with moderate to severe diarrhea (MSD) enrolled in the Global Enteric Multicenter Study (GEMS). We hypothesized that infection with each of these pathogens would induce distinct gut mucosal immune profiles indicative of disease etiology and severity. To test this hypothesis, innate and adaptive immune markers were measured in stools from children with diarrhea due to EPEC, Shigella , or other organisms and in children who had no diarrhea. Shigella- positive diarrhea evoked robust proinflammatory and T H 1/T H 2 cytokine responses compared to diarrhea caused by EPEC or other organisms, with the exception of interleukin 5 (IL-5), which was associated with EPEC infection. The presence of IL-1β, IL-4, IL-16, and tumor necrosis factor beta (TNF-β) was associated with the absence of dysentery. EPEC-positive diarrhea evoked high levels of IL-1β, vascular endothelial growth factor (VEGF), and IL-10. Granulocyte-macrophage colony-stimulating factor (GM-CSF) had opposing roles in disease severity, being associated with absence of diarrhea in EPEC-infected children and with dysenteric Shigella infection. High levels of antigen-specific antibodies were detected in the controls and children with Shigella without dysentery, which suggests a protective role against severe disease. In summary, this study identified distinct local immune responses associated with two clinically relevant diarrheagenic pathogens, Shigella and EPEC, in children and identified protective immune phenotypes that can inform the development of preventive measures. IMPORTANCE Shigella and enteropathogenic Escherichia coli are primary agents of moderate to severe diarrhea in children <5 years of age living in resource-poor countries. Repeated bouts of illness lead to lifelong health impairment and even death. Aiming to understand the local host immunity to these pathogens in relation to disease prognosis and to identify prophylaxis and therapeutic targets, we investigated innate and adaptive immune profiles in stools from children infected with EPEC with and without diarrhea, Shigella with and without dysentery, and controls in well characterized clinical samples obtained during the Global Enteric Multicenter Study. For the first time, we report pathogen-specific mucosal immune profiles associated with severity or absence of disease in children <5 years of age that can inform prevention and treatment efforts.

Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation efficiencies for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid-associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.

Shigella invasion plasmid antigen B (IpaB) plays an important role in causing shigellosis. While IpaB's protein structure, contribution to disease mechanism, and protective immunity against Shigella infection have been well studied, the significance of individual antigenic domains, especially at the N terminus, has not been systematically characterized. In an attempt to identify IpaB protein functional epitopes and to construct an optimized polyvalent multiepitope fusion antigen (MEFA) immunogen for development of a protein-based cross protective Shigella vaccine, in this study, we in silico identified immunodominant B-cell epitopes from the IpaB N terminus, fused each epitope to carrier protein CsaB (the major subunit of enterotoxigenic Escherichia coli CS4 adhesin) for epitope fusion proteins, immunized mice with each epitope fusion protein, examined IpaB-specific antibody responses, and assessed antibody functional activity against Shigella bacterial invasion. A total of 10 B-cell continuous epitopes were identified from IpaB N terminus, and after being fused to carrier protein CsaB, each epitope induced anti-IpaB IgG responses in the intramuscularly immunized mice. While in vitro antibody invasion inhibition assays demonstrated that antibodies derived from each identified epitope were functional, epitopes 1 (LAKILASTELGDNTIQAA), 2 (HSTSNILIPELKAPKSL), and 4 (QARQQKNLEFSDKI) induced antibodies to inhibit Shigella sonnei and Shigella flexneri invasion at levels similar to those of recombinant IpaB protein, suggesting that these three IpaB epitopes can be used potentially as IpaB-representing antigens to induce protective anti-IpaB antibodies and for construction of an epitope-based polyvalent MEFA protein immunogen for Shigella vaccine development. IMPORTANCE Currently, there are no effective measures for control or prevention of Shigella infection, the most common cause of diarrhea in children 3 to 5 years of age in developing countries. Challenges in developing Shigella vaccines include virulence heterogeneity among species and serotypes. To overcome virulence heterogeneity challenge and to develop a protein-based multivalent Shigella vaccine, we targeted a panel of virulence factors, including invasion plasmid antigens, identified functional antigenic domains or epitopes as representative antigens, and applied the novel epitope- and structure-based vaccinology platform multiepitope fusion antigen (MEFA) to integrate functional antigenic domains or epitopes into a backbone immunogen to produce a polyvalent immunogen for cross protective antibodies. Identification of functional IpaB epitopes from this study enhances our understanding of IpaB immunogenicity and allows us to directly utilize IpaB epitopes for construction of a cross protective polyvalent Shigella immunogen and to accelerate development of a protein-based Shigella vaccine.

Multiple drug-resistant (MDR) Shigella isolates have been reported worldwide. Between May 2017 and September 2018, 55 Shigella flexneri 2a isolates were collected from 3322 stool samples of 0-10-year-old outpatients with diarrhea at the Children's Hospital of Urumqi, China. All isolates were characterized using serotyping, antimicrobial susceptibility testing, and whole-genome sequencing. A total of 54 of 55 (98.2%) isolates exhibited MDR phenotypes and had accumulated multiple resistance determinants, particularly of fluoroquinolones and cephalosporins preferred for shigellosis treatment: point mutations in quinolone resistance-determining regions (QRDRs) of topoisomerases (GyrA (S83L, D87N) and ParC (S80I) [ n  = 9]; GyrA (S83L) and ParC (S80I) [ n  = 45]) and acquisition of qnrS1 ( n  = 3) and bla CTX-M ( n  = 8). Over 70% of isolates acquired two point mutations of GyrA (S83L) and ParC (S80I) in QRDRs and 11 highly resistant isolates accumulated three point mutations in QRDRs or acquired qnrS1 . Four S. flexneri 2a isolates from three single-nucleotide polymorphism clusters exhibited coresistance to ciprofloxacin, cefotaxime, or azithromycin (AZM), which are used as first- and second-line shigellosis treatment antimicrobials in clinics. Our data indicated that fluoroquinolones should be terminated in shigellosis treatment for outpatients in Urumqi. The transferable antimicrobial resistance determinants have been identified for third-generation cephalosporins and AZM. Novel strategies are urgently required for developing empirical medication to reduce the antimicrobial selective pressure and prevent dissemination of MDR S. flexneri 2a isolates.

Intractable functional constipation (IFC) is the most severe form of constipation, but its etiology has long been unknown. We hypothesized that IFC is caused by refractory infection by a pathogenic bacterium. Here, we isolated from patients with IFC a Shigella species - peristaltic contraction-inhibiting bacterium (PIB) - that significantly inhibited peristaltic contraction of the colon by production of docosapentenoic acid (DPA). PIB colonized mice for at least 6 months. Oral administration of PIB was sufficient to induce constipation, which was reversed by PIB-specific phages. A mutated PIB with reduced DPA was incapable of inhibiting colonic function and inducing constipation, suggesting that DPA produced by PIB was the key mediator of the genesis of constipation. PIBs were detected in stools of 56% (38 of 68) of the IFC patients, but not in those of non-IFC or healthy individuals (0 of 180). DPA levels in stools were elevated in 44.12% (30 of 68) of the IFC patients but none of the healthy volunteers (0 of 97). Our results suggest that Shigella sp. PIB may be the critical causative pathogen for IFC, and detection of fecal PIB plus DPA may be a reliable method for IFC diagnosis and classification.

Objectives: To evaluate a duplex droplet digital polymerase chain reaction (ddPCR) assay targeting Salmonella fimY and Shigella ipaH genes., Methods: The linear range, precision, analytical sensitivity, and analytical specificity of the ddPCR assay were analyzed. The ddPCR assay was compared with quantitative real-time PCR (qPCR) using 362 stool samples from 187 children with mild diarrhea and 175 children without diarrhea., Results: The duplex ddPCR assay showed good linearity in the range of 5.3 × 10 0 to 1.24 × 10 5 copies/reaction for Salmonella and 1.9 × 10 0 to 1.84 × 10 5 copies/reaction for Shigella. When analyzed with spiked stool samples, the limit of detection and limit of quantification were 550 and 5500 colony-forming units per mL of stool sample for Shigella, respectively, whereas both were 1.0 × 10 4 colony-forming units per mL of stool sample for Salmonella. Among 362 stool samples, more samples were detected as positive by ddPCR than by qPCR. Salmonella load was significantly higher in diarrheal samples than in non-diarrheal samples. Determined by receiver-operating characteristic analysis, the optimal cut-off value was 1.25 × 10 4 copies/mL of stool sample to distinguish between symptomatic and asymptomatic Salmonella infections., Conclusion: Salmonella and Shigella prevalence may have been underestimated in the past., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Viruses rely on hosts for their replication: thus, a critical step in the infection process is identifying a suitable host cell. Bacterial viruses, known as bacteriophages or phages, often use receptor binding proteins to discriminate between susceptible and non-susceptible hosts. By being able to evade predation, bacteria with modified or deleted receptor-encoding genes often undergo positive selection during growth in the presence of phage. Depending on the specific receptor(s) a phage uses, this may subsequently affect the bacteria's ability to form biofilms, its resistance to antibiotics, pathogenicity, or its phenotype in various environments. In this study, we characterize the interactions between two T4-like phages, Sf22 and KRT47, and their host receptor S. flexneri outer membrane protein C (OmpC). Results indicate that these phages use a variety of surface features on the protein, and that complete resistance most frequently occurs when hosts delete the ompC gene in full, encode premature stop codons to prevent OmpC synthesis, or eliminate specific regions encoding exterior loops., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This study was organized to investigate the prevalence, antibiotic and disinfectant resistance phenotypes and genotypes as well as plasmid profiles of Shigella species isolated from raw cow milk and milk products in Egypt. Genotypic analysis was performed to determine the presence of β-lactamase encoding genes (bla TEM , bla CTX-M , bla OXA-1 and bla SHV ), tet(A) and qacE∆. Forty-two (7%) Shigella isolates (S. dysenteriae, S. flexneri, and S. sonnei) were recovered, with S. dysenteriae as the predominant type. Antibiotic sensitivity tests showed that 71.4% of Shigella isolates were resistant to three or more antibiotic classes (multidrug-resistant). High resistance rates were observed against tetracyclines (100%), ampicillin, amoxicillin-clavulanate (90.5%, each) and cefaclor (66.7%), while no resistance was detected against imipenem, sulfamethoxazole/trimethoprim, and azithromycin. Disinfectant susceptibility test of Shigella isolates revealed resistance to phenolic compound (vanillic acid), while 85.7% of the Shigella isolates were resistant to benzalkonium chloride. Uniplex PCR analysis declared the existence of β-lactamase encoding genes (bla TEM in all isolates and bla CTX-M in 28.6% of isolates) and, tet(A) in all isolates and 85.7% of the isolates were positive for qacE∆1, while all isolates were negative for bla OXA-1 and bla SHV . All Shigella extended spectrum β-lactamases (ESBL) producers (12, 100%) were positive for the bla TEM , bla CTX-M , and qacE∆1 genes. Furthermore, plasmid profiling revealed seven distinct plasmid patterns (P1-P7), ranging from 1.26 to 33.61 kb, among all the Shigella strains; S. dysenteriae exhibited the greatest variance. The co-transfer of β-lactamase genes (bla TEM and bla CTX-M ) and qacE∆1 genes was observed by conjugation from all ESBL producers to a recipient strain. These findings indicate the emergence of Shigella species in Egypt that exhibited multi-resistance to either antibiotics (particularly ESBL producer strains) or disinfectants. Thus, the resistance of Shigella species should regularly be monitored and appropriate measures should be taken to manage this problem.

Purpose: Acute gastroenteritis (AGE) is caused by a wide range of pathogens. Culture methods for the detection of bacterial pathogens is time consuming and labour intensive. This study compared a same-day-to-result commercial molecular method using BD Max™ Enteric Bacterial Panel against conventional culture and laboratory-developed PCR assays (LDTs), and characterised the epidemiology of bacterial AGE in Singapore., Methodology: PCRs for Campylobacter spp., Salmonella spp., Shigella spp./Enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing E. coli (STEC)/Shigella dysenteriae were performed on the BD Max™ platform. Concurrent routine bacterial culture ("reference standard") was performed for Campylobacter, Salmonella, Shigella, Vibrio and Aeromonas spp. In the event of a discrepancy, an "expanded reference standard" (bacterial culture with LDT) was used., Results: There were 299 stool specimens in the study, with no bacterial pathogens detected in 190 samples (63.5%). The positive samples (n = 109,36.5%) were detected with Salmonella (n = 57,19.1%), Campylobacter (n = 28,9.4%), Vibrio parahaemolyticus (n = 6,2.0%), Shigella/EIEC (n = 6,2.0%), ETEC (n = 4,1.3%), STEC (n = 2,0.7%), Aeromonas (n = 2,0.7%), Plesiomonas shigelloides (n = 1,0.3%) and 3(1.0%) co-infections. Compared to the "expanded reference standard", conventional culture missed 38/112 (33.9%) pathogens. Conversely, testing by BD Max™ alone failed to detect 17 pathogens. BD Max™ reported seven (2.3%) false-positive results., Conclusions: BD Max™ increased the detection rate of bacterial AGE pathogens in the panel, but was limited by the absence of detection capability for Vibrio and Aeromonas spp., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Shigella flexneri is a major health burden in low- and middle-income countries, where it is a leading cause of mortality associated with diarrhoea in children, and shows an increasing incidence among travellers and men having sex with men. Like all Shigella spp., S. flexneri has evolved from commensal Escherichia coli following the acquisition of a large plasmid pINV, which contains genes essential for virulence. Current sequence typing schemes of Shigella are based on combinations of chromosomal genetic loci, since pINV-encoded virulence genes are often lost during growth in the laboratory, making these elements inappropriate for sequence typing. By performing comparative analysis of pINVs from S. flexneri strains isolated from different geographical regions and belonging to different serotypes, we found that in contrast to plasmid-encoded virulence genes, plasmid maintenance genes are highly stable pINV-encoded elements. For the first time, to our knowledge, we have developed a S. flexneri plasmid multilocus sequence typing (pMLST) method based on different combinations of alleles of the vapBC and yacAB toxin-antitoxin (TA) systems, and the parAB partitioning system. This enables typing of S. flexneri pINV plasmids into distinct 'virulence sequence types' (vSTs). Furthermore, the phylogenies of vST alleles and bacterial host core genomes suggests an intimate co-evolution of pINV with the chromosome of its bacterial host, consistent with previous findings. This work demonstrates the potential of plasmid maintenance loci as genetic characteristics to study as well as to trace the molecular phylogenesis of S. flexneri pINV and the phylogenetic relationship of this plasmid with its bacterial host.

Background: Shigella is a main cause of gastroenteritis and it is responsible for 5 to 10 % of diarrhea through the world. The aims of this study were to assess the antibiotic susceptibility pattern and the presence of 3 common virulence genes (sigA, virF, invE) of Shigella strains isolated from patients with gastroenteritis in Children's Medical Center Hospital, Tehran, Iran., Methods: Over a period of 15 months, all Shigella species collected from the patients with gastroenteritis were entered to the study. Susceptibility testing of all isolates towards different antibiotics was performed using the disk diffusion method and the prevalence of virulence genes was detected by polymerase chain reaction (PCR) technology., Results: Among a total of 183 Shigella strains, 128 Shigella sonnei (70%) and 55 S. flexneri (30%) were isolated. The resistance rate to the antibiotics in S. sonnei strains was higher than S. flexneri. The most sensitive antibiotics for S. flexneri strains were gentamicin (98%), amikacin (85%) and ciprofloxacin (82%), while high resistance rate to trimethoprim-sulfamethoxazole (96%), ampicillin (96%), nalidixic acid (64%) and cefotaxime (60%) was observed. The frequency of invE, virF and sigA gene in S. flexneri strains was 89 %, 93 % and 56 %, respectively; whereas they found in 93 %, 96 %, and 100 % of S. sonnei strains, respectively. SigA gene was identified significantly higher in the S. sonnei strains (100%). There was no significant difference between the presence of virF and invE genes among Shigella strains., Conclusion: The high presence of sigA gene in S. sonnei strains plays an important role in its pathogenesis, and the high frequency of invE and virF genes showed that this classical pathway regulating the expression of Shigella virulence factor genes could play a key role in the pathogenesis of this bacterium.

Enteroinvasive Escherichia coli (EIEC) is a diarrheagenic E. coli pathotype carrying a virulence plasmid that encodes a type III secretion system (TTSS) directly implicated in bacterial cell invasion. Since 2012, EIEC serotype O96:H19 has been recognized in Europe, Colombia, and most recently Uruguay. In addition to the invasion phenotype, the strains isolated from Colombian children with moderate-to-severe gastroenteritis had a strong biofilm formation phenotype, and as a result, they are referred to as biofilm-forming enteroinvasive E. coli (BF-EIEC). The objective of this study was to characterize the biofilm formation phenotype of the BF-EIEC O96:H19 strain 52.1 isolated from a child with moderate-to-severe gastroenteritis in Colombia. Random mutagenesis using Tn 5 transposons identified 100 mutants unable to form biofilm; 20 of those had mutations within the pgaABCD operon. Site-directed mutagenesis of pgaB and pgaC confirmed the importance of these genes in N -acetylglucosamine-mediated biofilm formation. Both biofilm formation and TTSS-mediated host cell invasion were associated with host cell damage on the basis of cytotoxic assays comparing the wild type, invasion gene mutants, and biofilm formation mutants. Multilocus sequence typing-based phylogenetic analysis showed that BF-EIEC strain 52.1 does not cluster with classic EIEC serotype strains. Instead, BF-EIEC strain 52.1 clusters with EIEC serotype O96:H19 strains described in Europe and Uruguay. In conclusion, BF-EIEC O96:H19, an emerging pathogen associated with moderate-to-severe acute gastroenteritis in children under 5 years of age in Colombia, invades cells and has a strong biofilm formation capability. Both phenotypes are independently associated with in vitro cell cytotoxicity, and they may explain, at least in part, the higher disease severity reported in Europe and Latin America. IMPORTANCE Enteroinvasive Escherichia coli (EIEC), a close relative of Shigella , is implicated in dysenteric diarrhea. EIEC pathogenicity involves cell invasion mediated by effector proteins delivered by a type III secretion system (TTSS) that disrupt the cell cytoskeleton. These proteins and the VirF global regulator are encoded by a large (>200 kb) invasion plasmid (pINV). This study reports an emergent EIEC possessing a cell invasion phenotype and a strong polysaccharide matrix-mediated biofilm formation phenotype. Both phenotypes contribute to host cell cytotoxicity in vitro and may contribute to the severe disease reported among children and adults in Europe and Latin America.

Aims: This study developed and evaluated a loop-mediated isothermal amplification (LAMP) assay to simply, rapidly and accurately identify Shigella flexneri serotypes 2 and Xv., Methods and Results: The LAMP assay based on the O-antigen synthesis and modification genes of S. flexneri including gtrII, gtrX, opt and wzx was developed. Its specificity and sensitivity were evaluated with 19 serotypes of S. flexneri and 96 other Shigella species and bacterial pathogens commonly found in stool samples. This LAMP assay was completed within 20 min at 61°C and could detect boiled DNA samples at concentrations as low as 1 pg/μl. The S. flexneri serotype LAMP assay exhibited 100% specificity for detecting 19 S. flexneri serotypes, no 96 strains of Shigella spp. and other bacterial pathogens. This LAMP assay was used to identify S. flexneri serotypes 2 and Xv from 299 S. flexneri strains isolated in China and results were consistent with that of slide agglutination and multiplex polymerase chain reaction results for the same isolates., Conclusions: This LAMP assay may facilitate rapid and reliable identifying S. flexneri serotypes 2 and Xv., Significance and Impact of Study: The present study was the first LAMP method for identifying serotypes of S. flexneri., (© 2021 Society for Applied Microbiology.)

Shigella species account for the second-leading cause of deaths due to diarrheal diseases among children of less than 5 years of age. The emergence of multi-drug-resistant Shigella isolates and the lack of availability of Shigella vaccines have led to the pertinence in the efforts made for the development of new therapeutic strategies against shigellosis. Consequently, designing small-interfering RNA (siRNA) candidates against such infectious agents represents a novel approach to propose new therapeutic candidates to curb the rampant rise of anti-microbial resistance in such pathogens. In this study, we analyzed 264 conserved sequences from 15 different conserved virulence genes of Shigella sp., through extensive rational validation using a plethora of first-generation and second-generation computational algorithms for siRNA designing. Fifty-eight siRNA candidates were obtained by using the first-generation algorithms, out of which only 38 siRNA candidates complied with the second-generation rules of siRNA designing. Further computational validation showed that 16 siRNA candidates were found to have a substantial functional efficiency, out of which 11 siRNA candidates were found to be non-immunogenic. Finally, three siRNA candidates exhibited a sterically feasible three-dimensional structure as exhibited by parameters of nucleic acid geometry such as: the probability of wrong sugar puckers, bad backbone confirmations, bad bonds, and bad angles being within the accepted threshold for stable tertiary structure. Although the findings of our study require further wet-lab validation and optimization for therapeutic use in the treatment of shigellosis, the computationally validated siRNA candidates are expected to suppress the expression of the virulence genes, namely: IpgD (siRNA 9) and OspB (siRNA 15 and siRNA 17) and thus act as a prospective tool in the RNA interference (RNAi) pathway. However, the findings of our study require further wet-lab validation and optimization for regular therapeutic use for treatment of shigellosis.

This study was to develop a multiplex fluorescent PCR for Shigella detection and species identification. Five primer pairs for Shigella detection and species identification were designed by Primer Premier 5.0. The multiplex fluorescent PCR was optimized by varying single parameter while other parameters were maintained. The multiplex fluorescent PCR assay could correctly detect Shigella and identify four Shigella species with a detection limits of 10 pg genomic DNA per reaction. Testing different strains and clinical samples confirmed the sensitivity and specificity of the multiplex fluorescent PCR. The newly developed multiplex fluorescent PCR assay is simple, sensitive and specific for Shigella detection and species identification. It has a potential to be used in routine Shigella detection and species identification in clinical laboratories., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Shigellosis is one of the major public health concerns in developing and low-income countries caused by four species of Shigella. There is an apparent need to develop rapid, cost-effective, sensitive and specific methods for differentiation of Shigella species to be used in outbreaks and health surveillance systems. We developed a sensitive and specific Fourier-transform infrared spectroscopy (FTIR) based method followed by principal component analysis (PCA) and hierarchical clustering analysis (HCA) assays to differentiate four species of Shigella isolates from stool samples. The FTIR based method was evaluated by differentiation of 91 Shigella species from each other in clinical samples using both gold standards (culture-based and agglutination methods) and developed FTIR assay; eventually, the sensitivity and specificity of the developed method were calculated. In summary, four distinct FTIR spectra associated with four species of Shigella were obtained with wide variations in three definite regions, including 1800-1550 cm -1 , 1550-1100 cm -1 , and 1100-800 cm -1 distinguish these species from each other. In this study, we found the FTIR method followed by PCA analysis with specificity, sensitivity, differentiation error and correct differentiation rate values of 100, 100, 0 and 100%, respectively, for identification and differentiation of all species of the Shigella in stool samples., (© 2022. The Author(s).)

Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections., Competing Interests: The authors have declared that no competing interests exist.

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations., (© 2022. The Author(s).)

Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay-"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user's bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user's need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~105 CFU/gm of stool for LT, STh, and STp and ~104 CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella., Competing Interests: The authors have declared that no competing interests exist.

The laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. Here, we study over 4000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes are grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognises the natural population structure of Shigella. By contrast, the serotyping scheme is affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will facilitate the transition from traditional phenotypic typing to routine whole-genome sequencing for the laboratory surveillance of Shigella infections., (© 2022. The Author(s).)

Molecular diagnostic methods improve the detection of Shigella , yet their ability to detect Shigella drug resistance on direct stool specimens is less clear. We tested 673 stool specimens from a Shigella treatment study in Bangladesh, including 154 culture-positive stool specimens and their paired Shigella isolates. We utilized a TaqMan array card that included quantitative PCR (qPCR) assays for 24 enteropathogens and 36 antimicrobial resistance (AMR) genes. Shigella was detected by culture in 23% of stool specimens (154/673), while qPCR detected Shigella at diarrhea-associated quantities in 49% (329/673; P < 0.05). qPCR for AMR genes on the Shigella isolates yielded >94% sensitivity and specificity compared with the phenotypic susceptibility results for azithromycin and ampicillin. The performance for trimethoprim-sulfamethoxazole susceptibility was less robust, and the assessment of ciprofloxacin was limited because most isolates were resistant. The detection of AMR genes in direct stool specimens generally yielded low specificities for predicting the resistance of the paired isolate, whereas the sensitivity and negative predictive values for predicting susceptibility were often higher. For example, the detection of ermB or mphA in stool yielded a specificity of 56% but a sensitivity of 91% and a negative predictive value of 91% versus the paired isolate's azithromycin resistance result. Patients who received azithromycin prior to presentation were universally culture negative (0/112); however, qPCR still detected Shigella at diarrhea-associated quantities in 34/112 (30%). In sum, molecular diagnostics on direct stool specimens greatly increase the diagnostic yield for Shigella , including in the setting of prior antibiotics. The molecular detection of drug resistance genes in direct stool specimens had low specificity for confirming resistance but could potentially "rule out" macrolide resistance.

Shigella species, a group of intracellular foodborne pathogens, are the main causes of bacillary dysentery and shigellosis in humans worldwide. It is essential to determine the species of Shigella in outbreaks and food safety surveillance systems. The available immunological and molecular methods for identifying Shigella species are relatively complicated, expensive and time-consuming. High resolution melting (HRM) assay is a rapid, cost-effective, and easy to perform PCR-based method that has recently been used for the differentiation of bacterial species. In this study, we designed and developed a PCR-HRM assay targeting rrsA gene to distinguish four species of 49 Shigella isolates from clinical and food samples and evaluated the sensitivity and specificity of the assay. The assay demonstrated a good analytical sensitivity with 0.01-0.1 ng of input DNA template and an analytical specificity of 100% to differentiate the Shigella species. The PCR-HRM assay also was able to identify the species of all 49 Shigella isolates from clinical and food samples correctly. Consequently, this rapid and user-friendly method demonstrated good sensitivity and specificity to differentiate species of the Shigella isolates from naturally contaminated samples and has the potential to be implemented in public health and food safety surveillance systems., (© 2022. The Author(s).)

Ribosomal RNA gene as a high-copy number nucleo-biomarker is extremely conserved among bacteria which limits its application to the discriminative detection approaches. We have developed a colorimetric isothermal amplification method called "single specific primer-LAMP (SSP-LAMP)" requiring only one specific primer for the amplification of the target and applied to the identification of the 16S rRNA gene in the Shigella genus. A region with high sequence homology in the genus and low homology with other bacteria was considered as the most appropriate. In that regard, a 23 bp sequence in the 16S rRNA gene of the genus was targeted based on the alignment of the gene with fifty-three closely related bacterial species, and a single specific primer along with five degenerate primers were designed. Using hydroxy-naphthol blue (HNB) as an indicator and gel electrophoresis, the proposed approach of SSP-LAMP was able to detect S. boydii, S. sonnei, S. flexneri and S. dysenteriae specifically while other species remained unidentified. The SSP-LAMP method could provide a rapid one-pot point-of-care method for molecular diagnostics of pathogens in many circumstances mainly samples with high genetic homogeneity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction. Bacterial dysentery is one of the greatest causes of morbidity and mortality worldwide. Campylobacter spp. and diarrhoeagenic Escherichia coli (DEC) are recognised as the most common causes of bacterial enteritis in developing countries including India. Hypothesis/Gap statement. Rapid and accurate identification of dysentery causing organisms using molecular methods is essential for better disease management, epidemiology and outbreak investigations. Aim. In view of the limited information available on the dysentery causing agents like Campylobacter spp., enterohemorrhagic E. coli (EHEC)/enteropathogenic E. coli (EPEC) and enteroinvasive E. coli (EIEC)/ Shigella in India, this study was undertaken to investigate the presence of these pathogens in human and poultry stool samples by molecular methods. Methodology. In total, 400 human stool samples and 128 poultry samples were studied. Microaerophilic culture along with real-time multiplex PCR with the targets specific to the genus Campylobacter , Campylobacter jejuni , Campylobacter coli , EHEC, EPEC and EIEC/ Shigella was performed. Further species confirmation was done using MALDI-TOF MS. Results. On microaerophilic culture, C. coli was isolated in one human sample and two C. jejuni and one C. fetus in poultry samples. On PCR analysis, among human stool samples, typical EPEC (42%) was predominantly seen followed by Campylobacter spp. (19%) and EIEC/ Shigella (10%). In contrast, Campylobacter spp. (41%) was predominant in poultry samples, followed by typical EPEC (26%) and EIEC/ Shigella (9%). Poly-infections with Campylobacter spp. and DEC were also observed among both sources. Conclusion. The present study documented the increased prevalence of Campylobacter spp. in humans compared with the results of previous studies from India. Typical EPEC was found to be predominant in children less than 5 years of age in this study. The high prevalence of coinfections in the current study indicates that a multiple aetiology of diarrhoea is common in our settings.

Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae , is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro . Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCE Shigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella 's major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium.

Iron is an essential element for Escherichia, Salmonella, and Shigella species. The acquisition of sufficient amounts of iron is difficult in many environments, including the intestinal tract, where these bacteria usually reside. Members of these genera have multiple iron transport systems to transport both ferrous and ferric iron. These include transporters for free ferrous iron, ferric iron associated with chelators, and heme. The numbers and types of transport systems in any species reflect the diversity of niches that it can inhabit. Many of the iron transport genes are found on mobile genetic elements or pathogenicity islands, and there is evidence of the spread of the genes among different species and pathotypes. This is notable among the pathogenic members of the genera in which iron transport systems acquired by horizontal gene transfer allow the bacteria to overcome host innate defenses that act to restrict the availability of iron to the pathogen. The need for iron is balanced by the need to avoid iron overload since excess iron is toxic to the cell. Genes for iron transport and metabolism are tightly regulated and respond to environmental cues, including iron availability, oxygen, and temperature. Master regulators, the iron sensor Fur and the Fur-regulated small RNA (sRNA) RyhB, coordinate the expression of iron transport and cellular metabolism genes in response to the availability of iron.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

The Shigella genus includes serious foodborne disease etiologic agents, with 4 species and 54 serotypes. Identification at species and serotype levels is a crucial task in microbiological laboratories. Nevertheless, the genetic similarity between Shigella spp. and Escherichia coli challenges the correct identification and serotyping of Shigella spp., with subsequent negative repercussions on surveillance, epidemiological investigations, and selection of appropriate treatments. For this purpose, multiple techniques have been developed historically ranging from phenotype-based methods and single or multilocus molecular techniques to whole-genome sequencing (WGS). To facilitate the selection of the most relevant method, we herein provide a global overview of historical and emerging identification and serotyping techniques with a particular focus on the WGS-based approaches. This review highlights the excellent discriminatory power of WGS to more accurately elucidate the epidemiology of Shigella spp., disclose novel promising genomic targets for surveillance methods, and validate previous well-established methods., (© 2021. Sociedade Brasileira de Microbiologia.)

Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. Differentiation of Shigella from EIEC is important for clinical diagnostic and epidemiological investigations. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli , making it difficult to find genetic markers to discriminate between Shigella and EIEC. In this study, we identified 10 Shigella clusters, seven EIEC clusters and 53 sporadic types of EIEC by examining over 17000 publicly available Shigella and EIEC genomes. We compared Shigella and EIEC accessory genomes to identify cluster-specific gene markers for the 17 clusters and 53 sporadic types. The cluster-specific gene markers showed 99.64% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella and EIEC serotype-specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired-end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70 and 99.74% cluster assignment accuracy for the assembled genomes and read mapping respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity of 99.40% for assembled genomes and 99.38% for read mapping for serotyping. The cluster-specific gene markers and our new serotyping tool, ShigEiFinder (installable package: https://github.com/LanLab/ShigEiFinder, online tool: https://mgtdb.unsw.edu.au/ShigEiFinder/), will be useful for epidemiological and diagnostic investigations.

Increasing resistance to first-line antibiotics used in the treatment of infections caused by Salmonella and Shigella species is emerging. Azithromycin presents a good alternative treatment option for Salmonella and Shigella infections. However, there are limited data regarding the susceptibility of azithromycin in Turkey. In this study, we aimed to evaluate the susceptibility of Salmonella and Shigella species to azithromycin, to determine and compare the minimum inhibitory concentration (MIC) values and disk diffusion zone diameters. In addition, susceptibility to meropenem and first-line antibiotic options in isolates was also investigated. A total of 170 Salmonella, 76 Shigella clinical isolates collected between 2014 and 2018 in our hospital were tested for their susceptibility to azithromycin, meropenem, ampicillin, pefloxacin, trimetoprim-sulfamethoxazole, ceftazidime, and cefotaxime. Isolates were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry. The isolates were confirmed and serotyped by the reference laboratory using the conventional slide agglutination method. Susceptibility of the isolates to azithromycin and other antibiotics was evaluated by Kirby-Bauer disk diffusion method. MIC values of azithromycin were determined by the reference broth microdilution method. Combined disk diffusion test was used for the detection of extended spectrum beta-lactamase (ESBL) production. Polymerase chain reaction was performed for macrolide and carbapenem resistance genes and the detected resistance genes were confirmed by sequencing. Of the 76 Shigella isolates tested, 64 (84.2%) were identified as Shigella sonnei, 10 (13.2%) as Shigella flexneri, one (1.3%) as Shigella boydii, and one (1.3%) as Shigella dysenteriae. Among the 170 Salmonella isolates, 131 (77%) were identified as Salmonella enteritidis, 11(6.5%) as Salmonella Typhimurium, 8 (4.7%) as Salmonella Kentucky, 5 (2.9%) as Salmonella Paratyphi B, 4 (2.4%) as Salmonella Infantis, 3 (1.8%) as Salmonella Cholerasuis, and 8 (4.7%) as other serovars (Salmonella Agona, Salmonella Dabou, Salmonella Gallinarum, Salmonella Hadar, Salmonella Muenchen, Salmonella Newport, Salmonella Paratyphi C, Salmonella Senftenberg), respectively. ESBL production was determined as 7.9% (6/76) in Shigella isolates and 2.9% (5/170) in Salmonella isolates. A carbapenem resistant S.Senftenberg isolate positive for the blaOXA-48 resistance gene was detected in our study. Meropenem MIC value of the isolate was detected as > 32 µg/ml with gradient diffusion test. Among all isolates, only one S.boydii isolate was detected as resistant to azithromycin with a MIC value of 128 µg/ml. The isolate was positive for the existence of mphA gene by PCR. In the disk diffusion test, azithromycin inhibition zone diameters were ≥ 12 mm in all of the tested isolates, except for the azithromycin-resistant isolate, and the azithromycin MICs were determined as ≤ 16 µg/ ml by broth microdilution. Increasing resistance to commonly used antibiotics in Salmonella and Shigella species is emerging. The detection of a carbapenem-resistant Salmonella isolate in our study indicates that the spread of carbapenem resistance to other Enterobacterales species may cause global problems. Antimicrobial susceptibility testing of azithromycin for Salmonella and Shigella species has been difficult to establish due to the lack of approval in vitro breakpoints for all species. Consequently, our data shows that azithromycin exhibits as a good alternative therapeutic choice for the treatment of gastrointestinal diseases caused by Salmonella and Shigella species. Further studies are needed to provide appropriate in vitro breakpoints supported by clinical data.

Background: Diarrheal disease is a leading cause of morbidity and mortality globally, especially in low- and middle-income countries. High-throughput and low-cost approaches to identify etiologic agents are needed to guide public health mitigation. Nanoliter-qPCR (nl-qPCR) is an attractive alternative to more expensive methods yet is nascent in application and without a proof-of-concept among hospitalized patients., Methods: A census-based study was conducted among diarrheal patients admitted at two government hospitals in rural Bangladesh during a diarrheal outbreak period. DNA was extracted from stool samples and assayed by nl-qPCR for common bacterial, protozoan, and helminth enteropathogens as the primary outcome., Results: A total of 961 patients were enrolled; stool samples were collected from 827 patients. Enteropathogens were detected in 69% of patient samples; More than one enteropathogen was detected in 32%. Enteropathogens most commonly detected were enteroaggregative Escherichia coli (26.0%), Shiga toxin-producing E.coli (18.3%), enterotoxigenic E. coli (15.5% heat stable toxin positive, 2.2% heat labile toxin positive), Shigella spp. (14.8%), and Vibrio cholerae (9.0%). Geospatial analysis revealed that the median number of pathogens per patient and the proportion of cases presenting with severe dehydration were greatest amongst patients residing closest to the study hospitals.", Conclusions: This study demonstrates a proof-of-concept for nl-qPCR as a high-throughput low-cost method for enteropathogen detection among hospitalized patients., Competing Interests: The authors have read the journal’s policy and have the following competing interests: EJN is associated with a patent on data collection software (Outbreak Responder, A Method, Apparatus and Computer Program Product for Personal Identification and Electronic Data Management) used in this study (Patent Publication Number 2020/0082921). The software development was funded by the National Institutes of Health (DP5OD019893), the University of Florida and Stanford University. EJN has no financial interest in the development and deployment of the software. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no other patents, products in development or marketed products associated with this research to declare. Development or marketed products associated with this research to declare.

Purpose of Review: To describe the impact of molecular diagnostics on our understanding of the burden and epidemiology of shigellosis in children in low-income and middle-income countries., Recent Findings: The incorporation of molecular diagnostics has led to a substantial increase in estimates of the burden of shigellosis and have allowed for further resolution of other aspects of Shigella epidemiology, including the clinical characteristics of shigellosis, the association between clinical and subclinical Shigella infection and linear growth shortfalls, protection after natural infection, duration of convalescent shedding, and host determinants of susceptibility., Summary: The increased sensitivity and precision afforded by molecular approaches has represented a major advance in our understanding of the epidemiology and burden of shigellosis in the settings of highest importance., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Species identification of Shigella isolates are so prominent for epidemiological studies and infection prevention strategies. We developed and evaluated RAPD and ERIC-PCR coupled with HRM for differentiation of non-dysenteriae Shigella species as potential alternative methods. After isolation of eighteen Shigella strains from faecal specimens collected from children under 2 years of age with diarrhea (n = 143), the species of the isolates were identified by slide agglutination assay. Also, species were identified using developed RAPD-PCR-HRM and ERIC-PCR-HRM techniques. Differentiation of the data sets was measured by principal component analysis as a dimension reduction method. Then, sensitivity and specificity of the methods were evaluated., Results: We found RAPD-PCR-HRM method with high sensitivity and specificity (100 and 85% respectively) to identify non-dysenteriae Shigella species in clinical specimens. However, sensitivity and specificity of ERIC-PCR-HRM were evaluated 33 and 46% respectively and significantly lower than that of RAPD-PCR-HRM assay. Regardless of inherent poor reproducibility of DNA fingerprinting-based methods, RAPD-PCR-HRM assay can be considered as a potential alternative method to identify non-dysenteriae species of Shigella in clinical specimens. As we observed in the current study, HRM technique is more rapid, inexpensive, and sensitive than gel electrophoresis method to characterize PCR amplicons., (© 2021. The Author(s).)

Motivation: Volcano plots are used to select the most interesting discoveries when too many discoveries remain after application of Benjamini-Hochberg's procedure (BH). The volcano plot suggests a double filtering procedure that selects features with both small adjusted $P$-value and large estimated effect size. Despite its popularity, this type of selection overlooks the fact that BH does not guarantee error control over filtered subsets of discoveries. Therefore the selected subset of features may include an inflated number of false discoveries., Results: In this paper, we illustrate the substantially inflated type I error rate of volcano plot selection with simulation experiments and RNA-seq data. In particular, we show that the feature with the largest estimated effect is a very likely false positive result. Next, we investigate two alternative approaches for multiple testing with double filtering that do not inflate the false discovery rate. Our procedure is implemented in an interactive web application and is publicly available., (© The Author(s) 2021. Published by Oxford University Press.)

Background: In high-income countries, shigellosis is mainly found in travellers to high-risk regions or in men who have sex with men (MSM). This study investigated the genomic characteristics and the features of antimicrobial resistance of MSM-associated Shigella flexneri and Shigella sonnei circulating in Barcelona, Spain, elucidating their connectivity with contemporaneous Shigella spp. from other countries., Methods: Antimicrobial susceptibility, whole-genome sequencing, genomic characterization and phylogenetic analysis were performed in MSM-associated Shigella spp. recovered from 2015 to 2019. Reference genomes of MSM-associated Shigella spp. were included for contextualization and to determine their connection with international outbreaks., Results: In total, 44 S. flexneri and 26 S. sonnei were identified among MSM. Overall, 80% showed resistance to azithromycin, 65.7% showed resistance to trimethoprim-sulphamethoxazole and 32.8% showed resistance to ciprofloxacin; 27.1% were resistant to all three antimicrobials. mphA and/or ermB, and qnrS and mutations in the quinolone resistance determining regions were found in the azithromycin- and ciprofloxacin-resistant isolates, respectively. Additionally, two isolates carried bla CTX-M-27. Single-nucleotide-polymorphism-based analysis revealed that the isolates were organized into different lineages, most of which were closely related to dominant MSM-associated lineages described previously in the UK and Australia., Conclusions: This study investigated the circulation of lineages of S. flexneri and S. sonnei among MSM in Spain that were mainly resistant to first-/second-line oral treatments, and closely related to dominant MSM-associated lineages described previously in the UK and Australia. These data reinforce the urgent need for the implementation of public health measures focusing on the early detection and prevention of transmission of this emerging pathogen, which is contributing to the antimicrobial resistance crisis in sexually transmitted infections., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Shigella and Salmonella cause serious problems in many subjects, including young children and the elderly, especially in developing countries. Chimeric proteins carrying immunogens increase immune response. In-silico tools are applied to design vaccine candidates. Invasion plasmid antigens D (ipaD) gene is one of the Shigella virulence factors. The N-terminal region of the IpaD plays a significant role in invading the host cell. Invasion protein H (invH) gene plays important role in bacterial adherence and entry into epithelial cells. A recombinant chimeric construct, containing IpaD and InvH was designed and used as a vaccine candidate against Shigella and Salmonella enteritidis. After bioinformatics assessments, the construct was designed, synthesized, and expressed in E.coli. Chimeric protein, IpaD, and InvH were purified with Ni-NTA chromatography. Purified proteins were confirmed with western blotting and then were injected into separate mice groups. The antibody titer was estimated with an enzyme-linked immunosorbent assay (ELISA). Mice were challenged with 10, 100, and 1000 LD50 of Salmonella, and the sereny test was performed for Shigella. The Codon adaptation index of the chimeric gene was increased to 0.84. Validation results showed that 97.9% of residues lie in the favored or additional allowed region of the Ramachandran plot. A significant antibody rise was observed in all test groups. The immunized mice with chimer and InvH could tolerate 100 LD50 of Salmonella. In the sereny test, the application of bacteria treated with immunized mice sera of both antigens showed no infection in Guinea pigs' eyes. The recombinant protein could protect animal models against Salmonella and Shigella and therefore can be considered as a suitable vaccine candidate against these two pathogens.

Background: Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials., Methods: We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score., Results: Compared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score., Conclusions: A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Shigellosis in men who have sex with men (MSM) is caused by multidrug resistant Shigellae, exhibiting resistance to antimicrobials including azithromycin, ciprofloxacin and more recently the third-generation cephalosporins. We sequenced four bla CTX-M-27 -positive MSM Shigella isolates (2018-20) using Oxford Nanopore Technologies; three S. sonnei (identified as two MSM clade 2, one MSM clade 5) and one S. flexneri 3a, to explore AMR context. All S. sonnei isolates harboured Tn7/Int2 chromosomal integrons, whereas S. flexneri 3a contained the Shigella Resistance Locus. All strains harboured IncFII pKSR100-like plasmids (67-83kbp); where present bla CTX-M-27 was located on these plasmids flanked by IS 26 and IS 903B , however bla CTX-M-27 was lost in S. flexneri 3a during storage between Illumina and Nanopore sequencing. IncFII AMR regions were mosaic and likely reorganised by IS 26 ; three of the four plasmids contained azithromycin-resistance genes erm(B ) and mph(A ) and one harboured the pKSR100 integron. Additionally, all S. sonnei isolates possessed a large IncB/O/K/Z plasmid, two of which carried aph(3')-Ib/aph(6)-Id/sul2 and tet(A ). Monitoring the transmission of mobile genetic elements with co-located AMR determinants is necessary to inform empirical treatment guidance and clinical management of MSM-associated shigellosis.

Escherichia coli is a ubiquitous bacterium that has been widely exposed to antibiotics over the last 70 years. It has adapted by acquiring different antibiotic-resistance genes (ARGs), the census of which we aim to characterize here. To do so, we analysed 70 301 E. coli genomes obtained from the EnteroBase database and detected 1 027 651 ARGs using the AMRFinder, Mustard and ResfinderFG ARG databases. We observed a strong phylogroup and clonal lineage specific distribution of some ARGs, supporting the argument for epistasis between ARGs and the strain genetic background. However, each phylogroup had ARGs conferring a similar antibiotic class resistance pattern, indicating phenotypic adaptive convergence. The G+C content or the type of ARG was not associated with the frequency of the ARG in the database. In addition, we identified ARGs from anaerobic, non- Proteobacteria bacteria in four genomes of E. coli , supporting the hypothesis that the transfer between anaerobic bacteria and E. coli can spontaneously occur but remains exceptional. In conclusion, we showed that phylum barrier and intra-species phylogenetic history are major drivers of the acquisition of a resistome in E. coli .

Background: Nontyphoid Salmonella and Shigella can cause gastroenteritis in humans. Ceftriaxone (CRO) has been used to treat their infection, however, development of CRO resistance are often associated with plasmid-mediated bla CMY. Here, we investigated the presence of plasmid-mediated ISEcp-1 tnpA-bla CMY-2 -blc-sugE and the role of these genes in regulation of CRO susceptibility in different hosts., Methods: 194 strains of Salmonella serovars and Shigella were tested for CRO susceptibility. Non-susceptibility strains were examined for plasmid-mediated ISEcp-1 tnpA-bla CMY-2 -blc-sugE by PCR amplification, Southern blot, and DNA sequencing. The plasmid profiles were determined by HindIII-digested restriction fragment length polymorphism (RFLP). Four recombinant plasmids with different genes from ISEcp-1 tnpA-bla CMY-2 -blc-sugE were constructed and then were transferred into Escherichia coli and different Salmonella serovars to evaluate the CRO susceptibility., Results: Among 20 CRO-nonsusceptible isolates of Salmonella Choleraesuis (5), S. Typhimurium (4), S. Mons (1), S. Stanley (2) and Shigella sonnei (8) with plasmid-mediated bla CMY-2, 19 isolates carried the ISEcp-1 tnpA-bla CMY-2 -blc-sugE and only one isolate with tnpA-bla CMY-2 . Transformation of these plasmids into E. coli pir116 produced multidrug resistance. Furthermore, PCR-RFLP analysis determined 5 different plasmid profiles and identical RFLP pattern between S. Typhimurium and S. sonnei. Transformation of the recombinant plasmids into E. coli and different Salmonella serovars resulted in phenotypes ranging from susceptible to resistant (especially inducible resistance) to CRO that were dependent on the genes, and host., Conclusion: The CRO susceptibility associated with the ISEcp-1 tnpA-bla CMY-2 -blc-sugE element is regulated positively by ISEcp-1 tnpA and SugE and negatively regulated by Blc and unknown species-dependent host factor(s)., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020. Published by Elsevier B.V.)

Globally, it is estimated that there are 2 billion cases of diarrhoeal disease each year, with 525,000 children under the age of 5 years, dying from diarrhoea. This also affects 1 in 5 people in the UK each year. Rapid diagnosis, appropriate treatment and infection control measures are, therefore, particularly important. Currently, Public Health Wales and England Microbiology Division test for five key bacterial gastrointestinal pathogens, i.e. Escherichia coli O157 (VTEC), Shigella dysenteriae, Salmonella spp., Campylobacter spp. and Clostridioides difficile. There is, however, a poor success rate with identification of these pathogens, leaving the patient at risk from untreated infections. This study has developed effective and reliable tools with a high positive outcome for diagnosis of diarrhoeal infection. The study blindly analysed 592 samples, with the most abundant species being Shigella sonnei at 15%, and the top genus Bacteroides at 26%. Campylobacter spp. had an abundance of 4%, Clostridium difficile 3%, and Salmonella spp. 0.2%. There were also significant differences in abundance at genus level, between the Flemish Gut project and diarrhoea samples, with respect to Shigella (0.2%) and Campylobacter (0.1%). The project introduced a novel Shigella spp. (Escherichia) threshold of 5.32% to determine (Escherichia) a healthy or unhealthy community. A DMBiome model was developed to integrate the 5.32% threshold of Shigella spp., the Public Health laboratory tested pathogens, and two emerging enteropathogens. The overall positive outcome was that 89% of all samples were diagnosed with diarrhoea infections, leaving 11% unknown.

Objective: Although oral methotrexate (MTX) remains the anchor drug for rheumatoid arthritis (RA), up to 50% of patients do not achieve a clinically adequate outcome. In addition, there is a lack of prognostic tools for treatment response prior to drug initiation. This study was undertaken to investigate whether interindividual differences in the human gut microbiome can aid in the prediction of MTX efficacy in new-onset RA., Methods: We performed 16S ribosomal RNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of drug-naive patients with new-onset RA (n = 26). Results were validated in an additional independent cohort (n = 21). To gain insight into potential microbial mechanisms, we conducted ex vivo experiments coupled with metabolomics analysis to evaluate the association between microbiome-driven MTX depletion and clinical response., Results: Our analysis revealed significant associations of the abundance of gut bacterial taxa and their genes with future clinical response (q < 0.05), including orthologs related to purine and MTX metabolism. Machine learning techniques were applied to the metagenomic data, resulting in a microbiome-based model that predicted lack of response to MTX in an independent group of patients. Finally, MTX levels remaining after ex vivo incubation with distal gut samples from pretreatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a possible direct effect of the gut microbiome on MTX metabolism and treatment outcomes., Conclusion: Taken together, these findings are the first step toward predicting lack of response to oral MTX in patients with new-onset RA and support the value of the gut microbiome as a possible prognostic tool and as a potential target in RA therapeutics., (© 2020, American College of Rheumatology.)

Increasing antimicrobial resistance and limited alternative treatments have led to fluoroquinolone-resistant Shigella strain inclusion on the WHO global priority pathogens list. In this study we characterized multiple Shigella isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1 .

Shigellosis is a diarrheal disease caused mainly by Shigella flexneri and Shigella sonnei Infection is thought to be largely self-limiting, with short- to medium-term and serotype-specific immunity provided following clearance. However, cases of men who have sex with men (MSM)-associated shigellosis have been reported where Shigella of the same serotype were serially sampled from individuals between 1 and 1,862 days apart, possibly due to persistent carriage or reinfection with the same serotype. Here, we investigate the accessory genome dynamics of MSM-associated S. flexneri and S. sonnei isolates serially sampled from individual patients at various days apart to shed light on the adaptation of these important pathogens during infection. We find that pairs likely associated with persistent infection/carriage and with a smaller single nucleotide polymorphism (SNP) distance, demonstrated significantly less variation in accessory genome content than pairs likely associated with reinfection, and with a greater SNP distance. We observed antimicrobial resistance acquisition during Shigella carriage, including the gain of an extended-spectrum beta-lactamase gene during carriage. Finally, we explored large chromosomal structural variations and rearrangements in seven (five chronic and two reinfection associated) pairs of S. flexneri 3a isolates from an MSM-associated epidemic sublineage, which revealed variations at several common regions across isolate pairs, mediated by insertion sequence elements and comprising a distinct predicted functional profile. This study provides insight on the variation of accessory genome dynamics and large structural genomic changes in Shigella during persistent infection/carriage. In addition, we have also created a complete reference genome and biobanked isolate of the globally important pathogen, S. flexneri 3a. IMPORTANCE Shigella spp. are Gram-negative bacteria that are the etiological agent of shigellosis, the second most common cause of diarrheal illness among children under the age of five in low-income countries. In high-income countries, shigellosis is also a sexually transmissible disease among men who have sex with men. Within the latter setting, we have captured prolonged and/or recurrent infection with shigellae of the same serotype, challenging the belief that Shigella infection is short lived and providing an early opportunity to study the evolution of the pathogen over the course of infection. Using this recently emerged transmission scenario, we comprehensively characterize the genomic changes that occur over the course of individual infection with Shigella and uncover a distinct functional profile of variable genomic regions, findings that have relevance for other Enterobacteriaceae ., (© Crown copyright 2021.)

Objective: The aim of this study was to investigate the genetic relatedness and antimicrobial resistance among Shigella species isolated from food and stool samples. Using cross sectional study method, Shigella spp. were isolated from food and clinical samples using culture-based, biochemical and serological methods. Antimicrobial susceptibility and genetic relatedness among the isolates were evaluated using disk diffusion and RAPD-PCR methods respectively., Results: The prevalence of Shigella spp. were 4.84 and 7.7% in food and stool samples respectively. All food isolates were Sh. sonnei. 91.42% of the Shigella stool isolates were Sh. sonnei. 62.5% of food isolates were resistant to tetracycline. 46.8, 50 and 65.8% of clinical isolates were resistant to imipenem, amikacin and azithromycin respectively. 50 and 85.7% of the food and clinical isolates respectively were MDR. Dendrogram generated by RAPD-PCR showed that the isolates from food and stool samples were categorized in a same group. Close genetic relatedness between MDR Shigella isolates from food and clinical samples indicate that foods can be considered as one of the main vehicles for transmission of MDR Shigella to human causing acute diseases. Survey of MDR Shigella among food and clinical samples is strongly suggested to be implemented.

Background: Numerous multiplex-PCR assays are now available in routine diagnostics but their clinical value is controversial if a clear association between clinical symptoms and the detection of a particular pathogen is missing. The objective of this work was to evaluate a multiplex-PCR assay for the diagnosis of traveller's diarrhoea (TD) in a case-control study and to assess the concordance with the BioFire® FilmArray® Gastrointestinal Panel., Methods: Stool samples from cases (n = 61) and controls (n = 30) were collected during travel and analysed by the GI-EB Screening assay (Seegene) in a case-control study. The concordance with the BioFire® FilmArray® Gastrointestinal Panel was expressed as the proportion of participants in which both tests agreed in the category "detected" and "not detected"., Results: None of the test-target organisms (Campylobacter spp., Clostridioides difficile toxin A/B, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli, E. coli O157, Shiga toxin-producing E. coli, Yersinia enterocolitica) was significantly associated with TD GI-EB Screening assay. The GI-EB Screening assay had an agreement with the BioFire® FilmArray® of 86.8-100%., Conclusion: The selection of test-target organisms included in the GI-EB Screening assay appears inappropriate for the diagnostic work-up of TD as none of the detected pathogens was associated with TD. The GI-EB Screening assay had a good concordance with BioFire® FilmArray®.

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.