Serotyping, MLST, and Core Genome MLST Analysis of Salmonella enterica From Different Sources in China During 2004–2019

Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004–2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China.

Comparación teórica entre técnicas fenotípicas y genotípicas utilizadas en la identificación de Listeria monocytogenes

Listeria monocytogenes es un patógeno ubicuo intracelular, causante de la Listeriosis, la cual se considera una enfermedad transmitida por alimentos (ETA). En la actualidad existe una creciente demanda de consumidores de productos alimenticios tratados mínimamente que pueden favorecer la proliferación de este microorganismo. Es necesario contar con programas de vigilancia que incluyan métodos fiables para la detección de este patógeno en casos de brotes epidémicos. Esta revisión bibliográfica compara las ventajas y desventajas de las técnicas fenotípicas y genotípicas utilizadas en la determinación de L. monocytogenes con el fin de definir la más adecuada que permita obtener resultados confiables y en el menor tiempo posible. Se realizó una búsqueda bibliográfica en bases de datos como Pubmed, Science Direct, Proquest y Ovid, en inglés y español, utilizando los siguientes descriptores: L. monocytogenes, molecular typing, diagnosis, PCR y bacterial typing techniques. Estos se combinaron de diferentes maneras para, finalmente, recopilar setenta artículos que cumplieron con los criterios de selección propuestos. Como resultado se presentan las técnicas de diagnóstico fenotípico y genotípico que representan una opción útil para el aislamiento e identificación de este patógeno a partir de diferentes orígenes. Las técnicas revisadas permiten la diferenciación entre especies patógenas y no patógenas, así como de serotipos y genotipos con base en la implementación de procedimientos cuya fundamentación puede diferir, pero que igualmente pueden ser complementarias.

Antimicrobial Resistance Profiling and Molecular Epidemiological Analysis of Extended Spectrum β-Lactamases Produced by Extraintestinal Invasive Escherichia coli Isolates From Ethiopia: The Presence of International High-Risk Clones ST131 and ST410 Revealed

The treatment of invasive Escherichia coli infections is a challenge because of the emergence and rapid spread of multidrug resistant strains. Particular problems are those strains that produce extended spectrum β-lactamases (ESBL’s). Although the global characterization of these enzymes is advanced, knowledge of their molecular basis among clinical E. coli isolates in Ethiopia is extremely limited. This study intends to address this knowledge gap. The study combines antimicrobial resistance profiling and molecular epidemiology of ESBL genes among 204 E. coli clinical isolates collected from patient urine, blood, and pus at four geographically distinct health facilities in Ethiopia. All isolates exhibited multidrug resistance, with extensive resistance to ampicillin and first to fourth line generation cephalosporins and sulfamethoxazole-trimethoprim and ciprofloxacin. Extended spectrum β-lactamase genes were detected in 189 strains, and all but one were positive for CTX-Ms β-lactamases. Genes encoding for the group-1 CTX-Ms enzymes were most prolific, and CTX-M-15 was the most common ESBL identified. Group-9 CTX-Ms including CTX-M-14 and CTX-27 were detected only in 12 isolates and SHV ESBL types were identified in just 8 isolates. Bacterial typing revealed a high amount of strains associated with the B2 phylogenetic group. Crucially, the international high risk clones ST131 and ST410 were among the sequence types identified. This first time study revealed a high prevalence of CTX-M type ESBL’s circulating among E. coli clinical isolates in Ethiopia. Critically, they are associated with multidrug resistance phenotypes and high-risk clones first characterized in other parts of the world.

Classification of Salmonella enterica of the (Para-)Typhoid Fever Group by Fourier-Transform Infrared (FTIR) Spectroscopy

Typhoidal and para-typhoidal Salmonella are major causes of bacteraemia in resource-limited countries. Diagnostic alternatives to laborious and resource-demanding serotyping are essential. Fourier transform infrared spectroscopy (FTIRS) is a rapidly developing and simple bacterial typing technology. In this study, we assessed the discriminatory power of the FTIRS-based IR Biotyper (Bruker Daltonik GmbH, Bremen, Germany), for the rapid and reliable identification of biochemically confirmed typhoid and paratyphoid fever-associated Salmonella isolates. In total, 359 isolates, comprising 30 S. Typhi, 23 S. Paratyphi A, 23 S. Paratyphi B, and 7 S. Paratyphi C, respectively and other phylogenetically closely related Salmonella serovars belonging to the serogroups O:2, O:4, O:7 and O:9 were tested. The strains were derived from clinical, environmental and food samples collected at different European sites. Applying artificial neural networks, specific automated classifiers were built to discriminate typhoidal serovars from non-typhoidal serovars within each of the four serogroups. The accuracy of the classifiers was 99.9%, 87.0%, 99.5% and 99.0% for Salmonella Typhi, Salmonella Paratyphi A, B and Salmonella Paratyphi C, respectively. The IR Biotyper is a promising tool for fast and reliable detection of typhoidal Salmonella. Hence, IR biotyping may serve as a suitable alternative to conventional approaches for surveillance and diagnostic purposes.

Discrimination of bacteria using whole organism fingerprinting: the utility of modern physicochemical techniques for bacterial typing

This review compares and contrasts MALDI-MS, FT-IR spectroscopy and Raman spectroscopy for whole organism fingerprinting and bacterial typing.

NGS-Based S. aureus Typing and Outbreak Analysis in Clinical Microbiology Laboratories: Lessons Learned From a Swiss-Wide Proficiency Test

Whole genome sequencing (WGS) enables high resolution typing of bacteria up to the single nucleotide polymorphism (SNP) level. WGS is used in clinical microbiology laboratories for infection control, molecular surveillance and outbreak analyses. Given the large palette of WGS reagents and bioinformatics tools, the Swiss clinical bacteriology community decided to conduct a ring trial (RT) to foster harmonization of NGS-based bacterial typing. The RT aimed at assessing methicillin-susceptible Staphylococcus aureus strain relatedness from WGS and epidemiological data. The RT was designed to disentangle the variability arising from differences in sample preparation, SNP calling and phylogenetic methods. Nine laboratories participated. The resulting phylogenetic tree and cluster identification were highly reproducible across the laboratories. Cluster interpretation was, however, more laboratory dependent, suggesting that an increased sharing of expertise across laboratories would contribute to further harmonization of practices. More detailed bioinformatic analyses unveiled that while similar clusters were found across laboratories, these were actually based on different sets of SNPs, differentially retained after sample preparation and SNP calling procedures. Despite this, the observed number of SNP differences between pairs of strains, an important criterion to determine strain relatedness given epidemiological information, was similar across pipelines for closely related strains when restricting SNP calls to a common core genome defined by S. aureus cgMLST schema. The lessons learned from this pilot study will serve the implementation of larger-scale RT, as a mean to have regular external quality assessments for laboratories performing WGS analyses in a clinical setting.

Harmonization of whole genome sequencing for outbreak surveillance of Enterobacteriaceae and Enterococci

Whole genome sequencing (WGS), is becoming the facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. We performed a three-center ring trial to assert if inter-laboratory harmonization of WGS is achievable, for this goal. To this end, a set of 30 bacterial isolates comprising of various species belonging to the Enterobacteriaceae and Enterococcus genera were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data was analysed by 1 centre using BioNumerics (6.7.3) for i) genotyping origin of replications & antimicrobial resistance genes, ii) core-genome (cgMLST) for E. coli and K. pneumoniae & whole-genome multi locus sequencing typing (wgMLST) for all species. Additionally, a split k-mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, only in 2/27 and 3/15 comparisons a discrepant allele was called between two genomes, for E. coli and K. pneumonia, respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0-11 SNPs (average 3.4). Furthermore, we demonstrate that using different de novo assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonisation of data processing surveillance of bacterial outbreaks. Summarizing, multi-center WGS for bacterial surveillance is achievable, but only if protocols are harmonized.

Implementation of MALDI-TOF Mass Spectrometry and Peak Analysis: Application to the Discrimination of Cryptococcus Species and Their Interspecies Hybrids

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of Flight) is a type of mass spectrometry (MS) that has been widely implemented for the rapid identification of microorganisms over the last decade. The accuracy and flexibility of this method has encouraged researchers to implement the analysis of protein spectra obtained by MALDI-TOF for the discrimination of close-related species and bacterial typing. In this study, a standardized methodology based on the detection of species-specific protein peaks from the spectra obtained with MALDI-TOF is described. The methodology was applied to a collection of Cryptococcus spp. (n=70) previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region. An expanded ad-hoc database was also built for their discrimination with MALDI-TOF. This approach did not allow the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of the PLS-DA and SVM algorithms in a two-step analysis allowed 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. Besides, PCA analysis prior to SVM provided 98.45% correct discrimination of the 3 analyzed species in a one-step analysis. The method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.

Novel algorithms for PFGE bacterial typing: Number of co-migrated DNA fragments, linking PFGE to WGS results and computer simulations for evaluation of PulseNet international typing protocols

BackgroundStandard protocols for Pulsed-field gel electrophoresis (PFGE) were adopted and being used in a global scale for surveillance of many bacterial food-borne diseases. Matched PFGE bands are considered regardless of co-migration of different DNA fragments. Molecular epidemiology is turning toward whole genome sequencing (WGS). Although, WGS results can be digested In-silico, PFGE and WGS data are being compared separately. We describe a new image analysis algorithm that enables identification of how many DNA fragments co-migrate during PFGE. We built a database that compare described PFGE results to in-silico obtained digestion models (from WGS). Reliability of the method was assessed in-silico using novel computer simulation approach. From WGS, 1,816 digestion model (DMs) were obtained as recommended by PulseNet international. Simulation codes were designed to predict PFGE profiles when DMs are separated at 5% PFGE resolution in addition to expected co-migration levels.ResultsPFGE simulation has shown that about 35% of DNA fragments co-migrate at 5% PFGE resolution. Similar result was obtained when wet-lab PFGE profiles were analyzed using image analysis algorithm mentioned earlier. When image analysis results were compared to DMs, results returned by geltowgs.uofk.edu database revealed reasonable relatedness to DMs. In terms of number of PFGE typable DNA fragments, 45,517 were typable (representing 46.54% out of 97,801). Previously mentioned typable fragments (in terms of typable sizes) comprised 91.24% of the sum of nucleotides of all chromosomes tested (7.24 billion bp). However, significant variations were shown within and between different digestion protocols.ConclusionsIdentification of co-migration levels will reveal the third dimension of PFGE profiles. This will provide a better way for evaluating isolate relationships. Linking old PFGE results to WGS by means of simulation demonstrated here will provide a chance to link millions of PFGE epidemiological data accumulated during the last 24 years to the new WGS era. Evaluation of population dynamics of pathogenic bacteria will be deeper through space and time. Selection of restriction enzymes for PFGE typing will have a powerful in-silico evaluation tool.