Utilizing novel Escherichia coli-specific conserved signature proteins for enhanced monitoring of recreational water quality.

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Genome-Wide Investigation Reveals Potential Therapeutic Targets in Shigella spp.

Shigella stands as a major contributor to bacterial dysentery worldwide scale, particularly in developing countries with inadequate sanitation and hygiene. The emergence of multidrug-resistant strains exacerbates the challenge of treating Shigella infections, particularly in regions where access to healthcare and alternative antibiotics is limited. Therefore, investigations on how bacteria evade antibiotics and eventually develop resistance could open new avenues for research to develop novel therapeutics. The aim of this study was to analyze whole genome sequence (WGS) of human pathogenic Shigella spp. to elucidate the antibiotic resistance genes (ARGs) and their mechanism of resistance, gene-drug interactions, protein-protein interactions, and functional pathways to screen potential therapeutic candidate(s). We comprehensively analyzed 45 WGS of Shigella, including S. flexneri (n = 17), S. dysenteriae (n = 14), S. boydii (n = 11), and S. sonnei (n = 13), through different bioinformatics tools. Evolutionary phylogenetic analysis showed three distinct clades among the circulating strains of Shigella worldwide, with less genomic diversity. In this study, 2,146 ARGs were predicted in 45 genomes (average 47.69 ARGs/genome), of which only 91 ARGs were found to be shared across the genomes. Majority of these ARGs conferred their resistance through antibiotic efflux pump (51.0%) followed by antibiotic target alteration (23%) and antibiotic target replacement (18%). We identified 13 hub proteins, of which four proteins (e.g., tolC, acrR, mdtA, and gyrA) were detected as potential hub proteins to be associated with antibiotic efflux pump and target alteration mechanisms. These hub proteins were significantly (p < 0.05) enriched in biological process, molecular function, and cellular components. Therefore, the finding of this study suggests that human pathogenic Shigella strains harbored a wide range of ARGs that confer resistance through antibiotic efflux pumps and antibiotic target modification mechanisms, which must be taken into account to devise and formulate treatment strategy against this pathogen. Moreover, the identified hub proteins could be exploited to design and develop novel therapeutics against MDR pathogens like Shigella.

Characterization of genotypes and antimicrobial resistance profiles of clinical isolates of Shigella from patients in the southern region of Iran.

BACKGROUND: Shigella spp., which are facultative anaerobic bacilli within the Enterobacteriaceae family, present a significant public health burden due to their role as prominent contributors to diarrheal diseases worldwide. A molecular analysis can facilitate the identification and assessment of outbreaks involving this bacterium. So, we aimed to investigate the antibiotic susceptibility pattern and clonal relatedness of clinical Shigella spp. isolates obtained from patients with diarrhea in Hormozgan province, South of Iran. METHODS: From 2019 to 2021, a cross-sectional investigation was conducted on 448 stool samples obtained from patients who were experiencing diarrhea, in the southern region of Iran. Shigella spp. isolates were identified based on biochemical and serological tests. All Shigella species were verified using species-specific polymerase chain reaction (PCR), followed by susceptibility testing to antimicrobial agents. Subsequently, genotyping of all Shigella species was conducted using ERIC-PCR. RESULTS: Out of a total of 448 stool samples, the presence of Shigella was detected in 62 cases, accounting for a prevalence rate of 13.84%. Among the identified isolates, the majority were attributed to S. flexneri, representing 53.23% of the cases. This was followed by S. sonnei at 24.19% and S. boydii at 22.58%. Notably, no instances of S. dysenteriae were found. The highest prevalence of Shigella isolates was observed in infants and children under the age of five. A significant proportion of the identified isolates demonstrated resistance to various antibiotics. Specifically, high resistance rates were noted for ampicillin (90.78%), piperacillin-tazobactam (87.1%), cefixime (83.87%), trimethoprim-sulfamethoxazole (83.87%), cefotaxime (82.26%), and ceftriaxone (80.65%). In addition, a substantial number (87.1%) of the isolates exhibited a multidrug-resistant (MDR) phenotype. Using the ERIC-PCR method, a total of 11 clusters and 6 distinct single types were identified among all the Shigella isolates. CONCLUSION: A notable occurrence of antibiotic-resistant Shigella species has been noted, with multi-drug resistant (MDR) strains presenting an increasing challenge for treating shigellosis worldwide, and this includes Iran. Techniques such as ERIC-PCR are useful for assessing the genetic variation and connections between Shigella strains, which indirectly contributes to understanding antimicrobial resistance patterns. Further research is needed to explore the specific correlation between resistance genes and ERIC genotyping patterns in Shigella strains.

The genomic epidemiology of shigellosis in South Africa.

Shigellosis, a leading cause of diarrhoeal mortality and morbidity globally, predominantly affects children under five years of age living in low- and middle-income countries. While whole genome sequence analysis (WGSA) has been effectively used to further our understanding of shigellosis epidemiology, antimicrobial resistance, and transmission, it has been under-utilised in sub-Saharan Africa. In this study, we applied WGSA to large sub-sample of surveillance isolates from South Africa, collected from 2011 to 2015, focussing on Shigella flexneri 2a and Shigella sonnei. We find each serotype is epidemiologically distinct. The four identified S. flexneri 2a clusters having distinct geographical distributions, and antimicrobial resistance (AMR) and virulence profiles, while the four sub-Clades of S. sonnei varied in virulence plasmid retention. Our results support serotype specific lifestyles as a driver for epidemiological differences, show AMR is not required for epidemiological success in S. flexneri, and that the HIV epidemic may have promoted Shigella population expansion.

Differential invasiveness & expression of antimicrobial peptides in Shigella serotypes.

Background & objectives: The study of Shigella pathogenesis at present is severely hampered by the lack of a relevant animal model that replicates human bacillary dysentery. Different Shigella serogroups cause varying severity of clinical illness. Ex vivo colonization of Shigella flexneri, S. dysenteriae and S. sonnei were characterized in human paediatric colonic pinch biopsies in the in vitro organ culture (IVOC) model to study the invasiveness of Shigella by gentamicin protection assay (GPA). Furthermore, the expression of antimicrobial peptides (AMPs) in response to different serotypes of Shigella was also studied in IVOC model. Methods: IVOC explants were inoculated with 109 colony forming units of different serotypes of Shigella and recovery of bacteria studied. Histopathological analysis was carried out to study inflammatory immune responses. GPA was done to elucidate the invasiveness of different serotypes of Shigella. Secretions of AMPs were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was performed to check the expression of AMPs and nuclear factor kappa B in IVOC explants. Results: After 24 h post-infection, the colon biopsies showed intense inflammatory reaction. In both IVOC and GPA, S. dysenteriae 1 was the most invasive as compared to S. flexneri and S. sonnei. S. sonnei was the least invasive. ELISA demonstrated that S. sonnei dampened the HBD (human ß-defensin)-2 responses whereas there was augmentation by S. dysenteriae and there was a modest but non-significant increase by S. flexneri. A modest increase in HBD-3 by S. sonnei and S. flexneri was observed but was not found to be significant. However, western blotting data showed upregulation of all AMPs by all serotypes. Western blotting is more sensitive than ELISA. Interpretation & conclusions: In the present study, differences in invasiveness and AMP production induced by different serotypes of Shigella were found. Human intestinal IVOC represents a model system to investigate early interaction between pathogenic bacteria and the human gut.

Modifying Escherichia coli to mimic Shigella for medical microbiology laboratory teaching: a new strategy to improve biosafety in class.

Introduction: Laboratory teaching of medical microbiology involves highly pathogenic microorganisms, thus posing potential biosafety risks to the students and the teacher. To address these risks, non/low-pathogenic microorganisms were modified to mimic highly pathogenic ones or highly pathogenic microorganisms were attenuated directly using the CRISPR/Cas9 technology. This study describes the modification of Escherichia coli DH5α to mimic Shigella and its evaluation as a safe alternative for medical laboratory teaching. Methods: To generate E. coli DH5α△FliC△tnaA2a, the tnaA and FliC genes in E. coli DH5α were knocked out using CRISPR/Cas9 technology; a plasmid bearing the O-antigen determinant of S. flexneri 2a was then constructed and transformed. Acid tolerance assays and guinea pig eye tests were used to assess the viability and pathogenicity, respectively. Questionnaires were used to analyze teaching effectiveness and the opinions of teachers and students. Results: The survey revealed that most teachers and students were inclined towards real-time laboratory classes than virtual classes or observation of plastic specimens. However, many students did not abide by the safety regulations, and most encountered potential biosafety hazards in the laboratory. E. coli DH5α△FliC△tnaA2a was biochemically and antigenically analogous to S. flexneri 2a and had lower resistance to acid than E. coli. There was no toxicity observed in guinea pigs. Most of teachers and students were unable to distinguish E. coli DH5α△FliC△tnaA2a from pure S. flexneri 2a in class. Students who used E. coli DH5α△FliC△tnaA2a in their practice had similar performance in simulated examinations compared to students who used real S. flexneri 2a, but significantly higher than the virtual experimental group. Discussion: This approach can be applied to other high-risk pathogenic microorganisms to reduce the potential biosafety risks in medical laboratory-based teaching and provide a new strategy for the development of experimental materials.

Invasion of HeLa Cells by Shigella Species Clinical Isolates Recovered from Pediatric Diarrhea.

Shigella is considered a major public health concern, especially for children younger than 5 years of age in developing countries. The pathogenicity of Shigella is a complex process that involves the interplay of multiple genes located on a large, unstable virulence plasmid as well as chromosomal pathogenicity islands. Since various factors (including virulence and antibiotic resistance genes) are associated with the severity and duration of shigellosis, in this article, we aim to evaluate whether the invasion of HeLa cells is affected by Shigella spp. isolates with different characteristics (including serogroups, virulence gene profiles, and antibiotic resistance patterns) recovered from pediatric patients in Tehran, Iran. Cell invasion ability of 10 Shigella isolates with different serogroups (Shigella flexneri and Shigella sonnei), gene profiling (virA, sen, ipgD, ipaD, ipaC, ipaB, and ipaH), and antibiotic resistance phenotyping (ampicillin, azithromycin, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefixime, cefotaxime, minocycline, and levofloxacin) were measured by plaque-forming assay in HeLa cell lines. The results show that all the selected Shigella spp. isolates recovered from pediatric patients were able to invade HeLa cells, but the total number and average size of plaques were different between the isolates. The higher invasion ability of S. flexneri isolates in HeLa cells compared to S. sonnei isolates was attributed to the presence of particular virulence genes; however, the role of each of these virulence factors remains to be determined.

Resistance in Enteric Shigella and nontyphoidal Salmonella : emerging concepts.

PURPOSE OF REVIEW: The emergence of globally resistant enteric Shigella and nontyphoidal Salmonella strains (NTS) has limited the selection of effective drugs, which has become a major challenge for the treatment of infections. The purpose of this review is to provide the current opinion on the antimicrobial-resistant enteric Shigella and nontyphoidal Salmonella . RECENT FINDINGS: Enteric Shigella and NTS are resistant to almost all classes of antimicrobials in recent years. Those with co-resistance to ciprofloxacin, azithromycin and ceftriaxone, the first-line antibiotics for the treatment of infectious diarrhoea have emerged worldwide. Some of them have caused interregional and international spread by travel, trade, MSM, and polluted water sources. Several strains have even developed resistance to colistin, the last-resort antibiotic used for treatment of multidrug-resistant Gram-negative bacteria infections. SUMMARY: The drug resistance of enteric Shigella and NTS is largely driven by the use of antibiotics and horizontal gene transfer of mobile genetic elements. These two species show various drug resistance patterns in different regions and serotypes. Hence treatment decisions for Shigella and Salmonella infections need to take into consideration prevalent antimicrobial drug resistance patterns. It is worth noting that the resistance genes such as blaCTX,mph, ermB , qnr and mcr , which can cause resistance to ciprofloxacin, cephalosporin, azithromycin and colistin are widespread because of transmission by IncFII, IncI1, IncI2 and IncB/O/K/Z plasmids. Therefore, continuous global monitoring of resistance in Shigella and Salmonella is imperative.

Occurrence of shigellosis in pediatric diarrheal patients in Chattogram, Bangladesh: A molecular based approach.

Shigellaa Gram-negative, non-motile bacillus, is the primary causative agent of the infectious disease shigellosis, which kills 1.1 million people worldwideevery year. The children under the age of five are primarily the victims of this disease. This study has been conducted to assess the prevalence of shigellosis through selective plating, biochemical test and conventional PCR assays, where the samples were collected from suspected diarrheoal patients. Invasive plasmid antigen H (ipaH) and O-antigenic rfc gene were used to identify Shigella spp. and S. flexneri respectively. For validation of these identification, PCR product of ipaH gene of a sample (Shigella flexneri MZS 191) has been sequenced and submitted to NCBI database (GenBank accession no- MW774908.1). Further this strain has been used as positive control. Out of 204, around 14.2% (n = 29)(P> 0.01) pediatric diarrheoal cases were screened as shigellosis. Another interesting finding was that most of shigellosis affected children were 7 months to 1 year (P> 0.01).The significance of this study lies in the analyses of the occurrenceand the molecular identification of Shigellaspp. and S. flexneri that can be utilized in improving the accurate identification and the treatment of the most severe and alarming shigellosis.

Acquisition of a large virulence plasmid (pINV) promoted temperature-dependent virulence and global dispersal of O96:H19 enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired. IMPORTANCE Enteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America.

No Isolate, No Problem: Using a Novel Insertion Sequence PCR to Link Rats to Human Shigellosis Cases in an Underserved Urban Community.

During an investigation into a cluster of Shigella flexneri serotype 2a cases in an underserved community, we assessed the relatedness of human and rat S. flexneri isolates utilizing a novel PCR targeting insertion sites (IS-PCR) of mobile elements in the Shigella genome characteristic of the cluster strain. Whole-genome sequences of S. flexneri (n = 50) associated with the cluster were analyzed. De novo genome assemblies were analyzed by a Geneious V10.2.6 motif search, and two unique IS were identified in all human Shigella sequences of the local cluster. Hydrolysis probe PCR assays were designed to detect these sequences consisting of forward and reverse primers to amplify across each insertion site and a hydrolysis probe spanning the insertion site. IS-PCR was performed for three Shigella PCR-positive culture-negative rat intestine specimens from this community. Both insertion sites were detected in the de novo genome assemblies of all clinical S. flexneri isolates (n = 50). Two of the three PCR-positive culture-negative rat samples were positive for both unique ISs identified in the human S. flexneri isolates, suggesting that the rat Shigella species strains were closely related to the human strains in the cluster. The cycle threshold (Ct) values were >35, indicating that the bacterial load was very low in the rat samples. Two unique IS were identified in clinical isolates from a community S. flexneri cluster. Both IS targets were identified in PCR-positive (Shigella spp.), culture-negative rat tissue and clinical isolates from humans, indicating relatedness. IMPORTANCE This article describes a novel molecular method to show relatedness between bacterial infections, which may not be able to grow in the laboratory due to treatment with antibiotics or for bacteria requiring unique conditions to grow well. Uniquely, we applied this technique to Shigella isolates from human cases associated with a local cluster in an underserved community, as well as rat samples from the same community. We believe that this novel approach can serve as a complementary method to support outbreak/cluster investigation for Shigella spp.

Concordance between Genotypic and Phenotypic Drug-Resistant Profiles of Shigella Isolates from Taiyuan City, Shanxi Province, China, 2005 to 2016.

Antimicrobial resistance in Shigella spp. is a global public health concern. In this study, the AMR phenotypic profiles of 10 kinds of antibiotics were compared with the genotypic profiles using genomic analysis of 218 Shigella isolates from Taiyuan City, Shanxi Province, China, 2005 to 2016. Core genome Multilocus Sequence Typing (cgMLST) based on the EnteroBase Escherichia/Shigella scheme was used to obtain the genetic relatedness of Shigella isolates. Multiple-drug resistance was observed in 96.79% Shigella spp., and the resistance to antimicrobial agents varied between S. flexneri and S. sonnei. The genotypic results correlated well with the phenotypic profiles with concordance rates of 96.42% and 94.50% in S. flexneri and S. sonnei isolates, respectively, from Taiyuan City, Shanxi Province. The sensitivity and specificity of the genotypic antimicrobial susceptibility testing (AST) were 97.56% and 95.34% for S. flexneri, and 95.65% and 93.31% for S. sonnei isolates, respectively. A discrepancy of genotypic and phenotypic AST results existed in some cephalosporin- and azithromycin-resistant Shigella isolates; there were no clear resistance patterns to predict ciprofloxacin resistance. There were major discrepancies between genotypic and phenotypic AST in the genotypically resistant but phenotypically susceptible isolates. The drug-resistance patterns and essential drug-resistance genes to predict the phenotypic drug-resistant profiles were the discrepancies between S. flexneri and S. sonnei isolates. Phylogenetic analysis showed that isolates of the same cluster but with different antibiotic-resistance gene patterns occurred because of the loss or gain of antibiotic-resistance genes located in the plasmids and multidrug-resistance islands. IMPORTANCE Antimicrobial resistance in Shigella spp. has become a global public health concern. In this study, we identified the antimicrobial susceptibility testing (AST) characteristics based on genomic sequences of 218 Shigella isolates and analyzed the correlation between genotypic and phenotypic antibiotic resistance profiles of Shigella spp., especially for fluoroquinolone, macrolides, and third-generation cephalosporins. Our results show that the genotypic results correlated with the phenotypic profiles with concordance rates of 96.42% and 94.50% in S. flexneri and S. sonnei isolates, respectively. The drug-resistance patterns and essential drug-resistance genes to predict the phenotypic drug-resistant profiles of S. flexneri and S. sonnei isolates in Taiyuan city were distinct. The discrepancy between genotypic and phenotypic AST was considerable in the genotypically resistant but phenotypically susceptible isolates. The information on drug resistance and resistance genes in this study can offer more details on the prevalence of drug resistance of Shigella spp.

Variable Region Sequences Influence 16S rRNA Performance.

16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain variable regions that can help distinguish different genera. However, intra-genus distinction using variable region homology is often impossible due to the high overall sequence identities among closely related species, even though some residues may be conserved within respective species. Using a computational method that included the allelic diversity within individual genomes, we discovered that certain Escherichia and Shigella species can be distinguished by a multi-allelic 16S rRNA variable region single nucleotide polymorphism (SNP). To evaluate the performance of 16S rRNAs with altered variable regions, we developed an in vivo system that measures the acceptance and distribution of variant 16S rRNAs into a large pool of natural versions supporting normal translation and growth. We found that 16S rRNAs containing evolutionarily disparate variable regions were underpopulated both in ribosomes and in active translation pools, even for an SNP. Overall, this study revealed that variable region sequences can substantially influence the performance of 16S rRNAs and that this biological constraint can be leveraged to justify refining taxonomic assignments of variable region sequence data. IMPORTANCE This study reevaluates the notion that 16S rRNA gene variable region sequences are uninformative for intra-genus classification and that single nucleotide variations within them have no consequence to strains that bear them. We demonstrated that the performance of 16S rRNAs in Escherichia coli can be negatively impacted by sequence changes in variable regions, even for single nucleotide changes that are native to closely related Escherichia and Shigella species; thus, biological performance is likely constraining the evolution of variable regions in bacteria. Further, the native nucleotide variations we tested occur in all strains of their respective species and across their multiple 16S rRNA gene copies, suggesting that these species evolved beyond what would be discerned from a consensus sequence comparison. Therefore, this work also reveals that the multiple 16S rRNA gene alleles found in most bacteria can provide more informative phylogenetic and taxonomic detail than a single reference allele.

Development and Validation of an Efficient Multiplex PCR Assay for Simultaneous Detection of Six Common Foodborne Pathogens and Hygiene Indicators.

Microbial contamination in foods could lead to illnesses and substantial losses in both food industry and public health sectors. Rapid detection of microbial hazards (i.e., pathogens, hygiene indicator microorganisms) can accelerate surveillance and diagnostic processes reducing transmission and minimizing undesirable consequences. This study developed a multiplex PCR (m-PCR) for the detection of six common foodborne pathogens and hygiene indicators using specific primers for uidA of Escherichia coli, stx2 of Escherichia coli O157:H7, invA of Salmonella spp., int of Shigella spp., ntrA of Klebsiella pneumoniae, and ail of Yersinia enterocolitica and Yersinia pseudotuberculosis. Sensitivity of the m-PCR was 100 fg or ∼20 bacterial cells. Each primer set amplified only the targeted strain, and specificity was demonstrated by lack of nonspecific bands with DNA from 12 other bacterial strains. Following ISO 16140-2:2016, the relative limit of detection of the m-PCR was comparable to that of the gold-standard method; however, the processing time was five times faster. The m-PCR was applied to detect the six pathogens in 100 natural samples (50 pork meat and 50 local fermented food samples) and compared to results of the gold-standard method. Positive cultures for Klebsiella, Salmonella, and E. coli were 66%, 82%, and 88%, respectively, of meat samples and 78%, 26%, and 56%, respectively, of fermented food samples. Escherichia coli O157:H7, Shigella, and Yersinia were not detected in any of the samples by both standard and m-PCR methods. The developed m-PCR assay showed comparable results with the traditional culture technique proving its rapid and reliable detection of six foodborne pathogens and hygiene indicators in food.

Phylogenetic trees of closely related bacterial species and subspecies based on frequencies of short nucleotide sequences.

Bacterial phylogenetic analyses are commonly performed to explore the evolutionary relationships among various bacterial species and genera based on their 16S rRNA gene sequences; however, these results are limited by mosaicism, intragenomic heterogeneity, and difficulties in distinguishing between related species. In this study, we aimed to perform genome-wide comparisons of different bacterial species, namely Escherichia coli, Shigella, Yersinia, Klebsiella, and Neisseria spp., based on their K-mer profiles to construct phylogenetic trees. Pentanucleotide frequency analyses (512 patterns of 5 nucleotides each) were performed to distinguish between highly similar species. Moreover, Escherichia albertii strains were clearly distinguished from E. coli and Shigella, despite being closely related to enterohemorrhagic E. coli in the phylogenetic tree. In addition, our phylogenetic tree of Ipomoea species based on pentamer frequency in chloroplast genomes was correlated with previously reported morphological similarities. Furthermore, a support vector machine clearly classified E. coli and Shigella genomes based on their pentanucleotide profiles. These results suggest that phylogenetic analyses based on penta- or hexamer profiles are a useful methodology for microbial phylogenetic studies. In addition, we introduced an R application, Phy5, which generates a phylogenetic tree based on genome-wide comparisons of pentamer profiles. The online version of Phy5 can be accessed at https://phy5.shinyapps.io/Phy5R/ and its command line version Phy5cli can be downloaded at https://github.com/YoshioNakano2021/phy5.

Whole-genome sequencing of Shigella for surveillance purposes shows (inter)national relatedness and multidrug resistance in isolates from men who have sex with men.

In the Netherlands, more than half of domestic shigellosis cases are among men who have sex with men (MSM), particularly in the Amsterdam region. However, there is limited insight into which Shigella strains circulate in the Netherlands. Our objective was to assess the added value of whole-genome sequencing (WGS)-based surveillance for Shigella. To this end, we determined the relatedness among Shigella spp. isolates from patients in the Amsterdam region, as well as in an international context, including antimicrobial resistance markers, using WGS. The following criteria were used: it should provide insight into (1) clustering of shigellosis cases and the affected population, (2) the extent of admixture of MSM-associated isolates with those from the broader population and (3) the presence of antimicrobial resistance. It should then lead to more opportunities for targeted control measures. For this study, Shigella isolates from three laboratories in the Amsterdam region obtained between February 2019 and October 2021 were subjected to Illumina WGS at the National Institute for Public Health and the Environment (RIVM). Raw data were quality-checked and assembled, the Shigella serotype was determined with ShigaTyper, and antimicrobial resistance markers were detected using ResFinder and PointFinder. For Shigella sonnei, subclades were determined using Mykrobe. Relatedness of isolates, including 21 international reference genomes, was assessed with core genome multilocus sequence typing. In total, 109 isolates were included, of which 27 were from females (25 %) and 66 were from males (61 %), with which the majority (n=48, 73 %) being from MSM. No information on sex was available for the remaining 16 cases. The WGS data for all isolates, comprising 55 S. sonnei, 52 Shigella flexneri, 1 Shigella boydii and 1 Shigella dysenteriae, met the quality criteria. In total, 14 clusters containing 51 isolates (49 %) were identified, with a median cluster size of 2.5 cases (range: 2-15). Nine out of 14 clusters were MSM-associated, and 8 clusters (57 %) were travel-related. Six of the MSM clusters were related to international reference genomes. The prevalence of antimicrobial resistance markers was higher among isolates from MSM than non-MSM patients, particularly for ciprofloxacin (89 vs 33 %) and azithromycin (58 vs 17 %). In conclusion, about half of Shigella spp. patients were part of a cluster, of which a substantial part were related to international reference genomes, particularly among MSM, and a high prevalence of antimicrobial resistance markers was found. These findings indicate widespread international circulation of Shigella spp., particularly among MSM, with multidrug resistance that hampers treatment of patients. Moreover, the results of this study led to the implementation of a national WGS-based laboratory surveillance programme for Shigella spp. that started in April 2022.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical Shigella and Salmonella isolates in Belgium.

For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated. The combination with long-read sequencing offers a potential solution, as it allows hybrid assemblies. Nevertheless, its use in surveillance has so far been limited. This study aimed to demonstrate its added value for AMR surveillance based on a case study of extended-spectrum beta-lactamases (ESBLs). ESBL genes have been reported to occur also on plasmids. To gain insight into the diversity and genetic context of ESBL genes detected in clinical isolates received by the Belgian National Reference Center between 2013 and 2018, 100 ESBL-producing Shigella and 31 ESBL-producing Salmonella were sequenced with MiSeq and a representative selection of 20 Shigella and six Salmonella isolates additionally with MinION technology, allowing hybrid assembly. The bla CTX-M-15 gene was found to be responsible for a rapid rise in the ESBL Shigella phenotype from 2017. This gene was mostly detected on multi-resistance-carrying IncFII plasmids. Based on clustering, these plasmids were determined to be distinct from the circulating plasmids before 2017. They were spread to different Shigella species and within Shigella sonnei between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing bla CTX-M-27 for which only clonal expansion occurred. Matches of up to 99 % to plasmids of various bacterial hosts from all over the world were found, but global alignments indicated that direct or recent ESBL-plasmid transfers did not occur. It is most likely that travellers introduced these in Belgium and subsequently spread them domestically. However, a clear link to a specific country could not be made. Moreover, integration of bla CTX-M in the chromosome of two Shigella isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in Salmonella, ESBL genes were only found on plasmids, of which bla CTX-M-55 and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical Shigella and Salmonella isolates. The hybrid assembly data allowed us to check the accuracy of plasmid prediction tools. MOB-suite showed the highest accuracy. However, these tools cannot replace the accuracy of long-read and hybrid assemblies. This study illustrates the added value of hybrid assemblies for AMR surveillance and shows that a strategy where even just representative isolates of a collection used for hybrid assemblies could improve international AMR surveillance as it allows plasmid tracking.