MALDI-TOF MS Using a Custom-Made Database, Biomarker Assignment, or Mathematical Classifiers Does Not Differentiate Shigella spp. and Escherichia coli

Shigella spp. and E. coli are closely related and cannot be distinguished using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) with commercially available databases. Here, three alternative approaches using MALDI-TOF MS to identify and distinguish Shigella spp., E. coli, and its pathotype EIEC were explored and evaluated using spectra of 456 Shigella spp., 42 E. coli, and 61 EIEC isolates. Identification with a custom-made database resulted in >94% Shigella identified at the genus level and >91% S. sonnei and S. flexneri at the species level, but the distinction of S. dysenteriae, S. boydii, and E. coli was poor. With biomarker assignment, 98% S. sonnei isolates were correctly identified, although specificity was low. Discriminating markers for S. dysenteriae, S. boydii, and E. coli were not assigned at all. Classification models using machine learning correctly identified Shigella in 96% of isolates, but most E. coli isolates were also assigned to Shigella. None of the proposed alternative approaches were suitable for clinical diagnostics for identifying Shigella spp., E. coli, and EIEC, reflecting their relatedness and taxonomical classification. We suggest the use of MALDI-TOF MS for the identification of the Shigella spp./E. coli complex, but other tests should be used for distinction

Genome-wide association studies of Shigella spp. and Enteroinvasive Escherichia coli isolates demonstrate an absence of genetic markers for prediction of disease severity

BACKGROUND: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control. RESULTS: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used. CONCLUSIONS: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC

A Multifactorial Approach for Surveillance of Shigella spp. and Entero-Invasive Escherichia coli Is Important for Detecting (Inter)national Clusters

Shigella spp. and entero-invasive Escherichia coli (EIEC) can cause mild diarrhea to dysentery. In Netherlands, although shigellosis is a notifiable disease, there is no laboratory surveillance for Shigella spp. and EIEC in place. Consequently, the population structure for circulating Shigella spp. and EIEC isolates is not known. This study describes the phenotypic and serological characteristics, the phenotypic and genetic antimicrobial resistance (AMR) profiles, the virulence gene profiles, the classic multi-locus sequence types (MLST) and core genome (cg)MLST types, and the epidemiology of 414 Shigella spp. and EIEC isolates collected during a cross-sectional study in Netherlands in 2016 and 2017. S. sonnei (56%), S. flexneri (25%), and EIEC (15%) were detected predominantly in Netherlands, of which the EIEC isolates were most diverse according to their phenotypical profile, O-types, MLST types, and cgMLST clades. Virulence gene profiling showed that none of the isolates harbored Shiga toxin genes. Most S. flexneri and EIEC isolates possessed nearly all virulence genes examined, while these genes were only detected in approximately half of the S. sonnei isolates, probably due to loss of the large invasion plasmid upon subculturing. Phenotypical resistance correlated well with the resistant genotype, except for the genes involved in resistance to aminoglycosides. A substantial part of the characterized isolates was resistant to antimicrobials advised for treatment, i.e., 73% was phenotypically resistant to co-trimoxazole and 19% to ciprofloxacin. AMR was particularly observed in isolates from male patients who had sex with men (MSM) or from patients that had traveled to Asia. Furthermore, isolates related to international clusters were also circulating in Netherlands. Travel-related isolates formed clusters with isolates from patients without travel history, indicating their emergence into the Dutch population. In conclusion, laboratory surveillance using whole genome sequencing as high-resolution typing technique and for genetic characterization of isolates complements the current epidemiological surveillance, as the latter is not sufficient to detect all (inter)national clusters, emphasizing the importance of multifactorial public health approaches

Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella species and Entero-invasive Escherichia coli in the Netherlands

An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coil (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic methods and a sample panel, consisting of DNA-extracts and spiked stool samples with different concentrations of Shigella flexneri, was provided to each MML The results of the trial showed an enormous variety in culture-dependent and molecular diagnostic techniques currently used among MMLs. Despite the various molecular procedures, 15 out of 16 MMLs were able to detect Shigella species or EIEC in all the samples provided, showing that the diversity of methods has no effect on the qualitative detection of Shigella flexneri. In contrast to semi quantitative analysis, the minimum and maximum values per sample differed by approximately five threshold cycles (Ct-value) between the MMLs included in the study. This indicates that defining a uniform Ct-value cut-off for notification to health authorities is not advisable. (C) 2016 Elsevier B.V. All rights reserved

Genome-wide association studies of Shigella spp. and Enteroinvasive Escherichia coli isolates demonstrate an absence of genetic markers for prediction of disease severity

BACKGROUND: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control. RESULTS: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used. CONCLUSIONS: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC

Evaluation of 5 '-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools

5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with reference strains of E. coli (n=40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx(1c), stx(1d), and stx(2f)), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log(10) and 3.42 to 4.63 log(10) CFU/g of stool for 5'-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation o

Evaluation of a culture dependent algorithm and a molecular algorithm for identification of Shigella spp., Escherichia coli, and enteroinvasive E. coli (EIEC).

Identification of Shigella spp., Escherichia coli and enteroinvasive E. coli is challenging, because of their close relatedness. Distinction is vital, as infections with Shigella spp. are under surveillance of health authorities, in contrast to EIEC infections. In this study, a culture dependent identification algorithm and a molecular identification algorithm were evaluated. Discrepancies between the two algorithms and original identification were assessed using Whole Genome Sequencing (WGS). After discrepancy analysis, with the molecular algorithm, 100% of the evaluated isolates were identified in concordance with original identification. However, the resolution for certain serotypes was lower than previously described methods and lower than the culture dependent algorithm. Although, the resolution of the culture dependent algorithm is high, 100% of non-invasive E. coli, S. sonnei, S. dysenteriae, 93% of S. boydii and EIEC and 85% of S. flexneri were identified in concordance with the original identification. Discrepancy analysis using WGS was able to confirm one of the used algorithms in four discrepant results. However, it failed to clarify three other discrepant results as it added yet another identification. Both proposed algorithms performed well for the identification of Shigella spp and EIEC, and are applicable in low-resource settings in contrast to earlier described methods that require WGS for daily diagnostics. Evaluation of the algorithms showed that both algorithms are capable of identifying Shigella species and EIEC isolates. The molecular algorithm is more applicable in clinical diagnostics for fast and accurate screening, while the culture dependent algorithm is more suitable for reference laboratories to identify Shigella spp. and EIEC up to serotype level

Prevalence and Risk Factors for Colonization With Extended-Spectrum Cephalosporin-Resistant Escherichia coli in Children Attending Daycare Centers: A Cohort Study in the Netherlands

BACKGROUND: The purpose of this study was to determine the prevalence and risk factors for colonization with extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli in daycare center (DCC)-attending children. METHODS: This is a prospective cohort study including 44 DCCs in the Netherlands, combining DCC characteristics and monthly collected stool samples from their attendees, and was performed in 2010-2012. During a 22-month study period, 852 stool samples were collected and screened for ESC-R E coli. Risk factors were studied using logistic regression analysis. RESULTS: In DCC-attending children (<4 years old), the overall prevalence of ESC-R E coli was 4.5%, and it was 8% in <1-year-old attendees. Among the 38 children carrying ESC-R E coli, the most common types were blaCMY-2 (26%), blaCTX-M-1 (16%), and chromosomal AmpC type 3 promoter mutants (13%). Extended-spectrum cephalosporin-resistant E coli was less common in DCCs where stricter hygiene protocols were enforced, eg, not allowing ill children to enter the DCC (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.14-0.84), performing extra checks on handwashing of ill children (OR, 0.42; 95% CI, 0.20-0.87), and reporting suspected outbreaks to local health authorities (OR, 0.27; 95% CI, 0.11-0.69). CONCLUSIONS: The distribution of ESC-R E coli types in DCCs differs from that of the general population. Extended-spectrum cephalosporin-resistant E coli carriage in DCC-attending children is associated with the hygiene policies enforced in the DCC. Although our results are not conclusive enough to change current DCC practice beyond ensuring compliance with standing policies, they generated hypotheses and defined the degree of ESC resistance among DCC attendees, which may influence empiric antibiotic therapy choices, and tracked the increasing trend in ESC resistance

Incidence, clinical implications and impact on public health of infections with Shigella spp. and entero-invasive Escherichia coli (EIEC): results of a multicenter cross-sectional study in the Netherlands during 2016-2017.

Background: Shigella spp. and entero-invasive E. coli (EIEC) use the same invasive mechanism to cause diarrheal diseases. Public health regulations apply only to Shigella spp. infections, but are hampered by the lack of simple methods to distinguish them from EIEC. In the last decades, molecular methods for detecting Shigella spp. and EIEC were implemented in medical microbiological laboratories (MMLs). However, shigellosis cases identified with molecular techniques alone are not notifiable in most countries. Our study investigates the impact of EIEC versus Shigella spp. infections and molecular diagnosed shigellosis versus culture confirmed shigellosis for re-examination of the rationale for the current public health regulations. Methods: In this multicenter cross-sectional study, fecal samples of patients suspected for gastro-enteritis, referred to 15 MMLs in the Netherlands, were screened by PCR for Shigella spp. or EIEC. Samples were cultured to discriminate between the two pathogens. We compared risk factors, symptoms, severity of disease, secondary infections and socio-economic consequences for (i) culture-confirmed Shigella spp. versus culture-confirmed EIEC cases (ii) culture positive versus PCR positive only shigellosis cases. Results: In 2016-2017, 777 PCR positive fecal samples with patient data were included, 254 of these were culture-confirmed shigellosis cases and 32 were culture-confirmed EIEC cases. EIEC cases were more likely to report ingestion of contaminated food and were less likely to be men who have sex with men (MSM). Both pathogens were shown to cause serious disease although differences in specific symptoms were observed. Culture-negative but PCR positive cases were more likely report travel or ingestion of contaminated food and were less likely to be MSM than culture-positive cases. Culture-negative cases were more likely to suffer from multiple symptoms. No differences in degree of secondary infections were observed between Shigella spp. and EIEC, and culture-negative and culture-positive cases. Conclusions: No convincing evidence was found to support the current guidelines that employs different measures based on species or detection method. Therefore, culture and molecular detection methods for Shigella spp. and EIEC should be considered equivalent for case definition and public health regulations regarding shigellosis. Differences were found regarding risks factors, indicating that different prevention strategies may be required