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

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

<p>Abstract</p> <p>Background</p> <p>The genus <it>Brucella </it>contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays.</p> <p>Results</p> <p>In this study, the accurate identification of <it>Brucella </it>species using MALDI-TOF-MS was achieved by constructing a <it>Brucella </it>reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from <it>Brucella </it>species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for <it>Brucella </it>species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and <it>B. suis </it>biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for <it>Brucella</it>, even minimal genomic differences between these serovars translate to specific proteomic differences.</p> <p>Conclusions</p> <p>MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.</p

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

Surveillance of diphtheria in the Netherlands between 2000–2021: cutaneous diphtheria supersedes the respiratory form

Abstract Background Diphtheria is a severe respiratory or cutaneous infectious disease, caused by exotoxin producing Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Diphtheria is once again prevalent due to breakdown of immunisation programmes, social disruption and unrest. Aim This study describes the notified diphtheria cases in the Netherlands between 2000–2021 and isolates that were sent to the National Institute for Public Health and the Environment (RIVM). Methods File investigation was performed including all notified cases and isolates of C. diphtheriae, C. ulcerans and C. pseudotuberculosis that were tested for toxin production using a toxin-PCR and Elek test. An exploratory review was performed to understand transmission in populations with a high vaccination uptake. Results Eighteen diphtheria notifications were made with confirmed toxigenic C. diphtheriae (n = 9) or ulcerans (n = 9) between 2000 and 2021. Seventeen (94.4%) presented with a cutaneous infection. All cases with a suspected source abroad (n = 8) concerned infection with C. diphtheriae. In contrast, 9/10 cases infected in the Netherlands were caused by C. ulcerans, a zoonosis. Secondary transmission was not reported. Isolates of C. ulcerans sent to the RIVM produced more often the diphtheria exotoxin (11/31; 35%) than C. diphtheriae (7/89; 7.9%). Conclusion Both human-to-human transmission of C. diphtheriae and animal-to-human transmission of C. ulcerans rarely occurs in the Netherlands. Cases mainly present with a cutaneous infection. Travel-related cases remain a risk for transmission to populations with low vaccination coverage, highlighting the importance of immunization and diphtheria control measures

Molecular Characterization of Capnocytophaga canimorsus and Other Canine Capnocytophaga spp. and Assessment by PCR of Their Frequencies in Dogs ▿ †

Capnocytophaga canimorsus can be a virulent pathogen, whereas C. cynodegmi is of low virulence. Heterogeneity within these species, their frequency in dogs, and pathogenicity factors are largely unknown. Strains from blood cultures from patients presumptively identified as C. canimorsus (n = 25) and as C. cynodegmi by rrs analysis (n = 4), blood cultures from dogs (n = 8), blood cultures from cats (n = 2), and cultures from swabs from dog mouths (n = 53) were analyzed. PCR-restriction fragment length polymorphism (PCR-RFLP), a species-specific PCR on rpoB, and rrs sequencing were used. All 29 strains from human blood cultures could be grouped into three PCR-RFLP types. One included the C. canimorsus type strain, and the other types were closely related. Two canine strains were C. canimorsus and grouped into the least common RLFP pattern group. Five were C. cynodegmi and clustered with the reference strain. One canine and both feline strains were distinct. Four human strains that presumptively had been identified as C. cynodegmi by RNA gene sequence analysis clustered with the C. canimorsus strains by both PCR-RFLP and the sequence-specific PCR of the rpoB gene. C. canimorsus DNA was present in 73% (range, 61 to 85%) of dogs' mouths, and C. cynodegmi DNA was present in 96% (range, 94 to 100%) of dogs' mouths. As defined by rpoB PCR-RFLP and by PCRs using specific primers, all strains from human blood were C. canimorsus. The sequencing of rrs genes suggested the presence of different gene copies in a few strains, indicating that the method is less appropriate for species identification. Both species are present in the majority of dogs. Additional Capnocytophaga species occur in dogs' and cats' mouths

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

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Indigenous Infection with Francisella tularensis holarctica in The Netherlands

We report here the first case of indigenous tularemia detected in The Netherlands, a nonendemic country, since 1953. Whole genome DNA sequence analysis assigned the isolate BD11-00177 to the genomic group B.FTNF002-00, which previously has been exclusively reported from Spain, France, Italy, Switzerland, and Germany. The patient had not been abroad for years, which implies that this is an indigenous infection. The current case might predict an upcoming distribution of Francisella tularensis holarctica genomic group B.FTNF002-00 in Europe