Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme.

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants' opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe.

Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans.

Multilocus sequence typing of the goose pathogen Mycoplasma anserisalpingitidis.

Mycoplasma anserisalpingitidis infection is associated with the inflammation of the genital tract and cloaca, embryo lethality, and decreased egg production in geese, leading to serious economic losses. M. anserisalpingitidis has been detected mainly in Central and Eastern Europe, especially in Hungary, but the pathogen was identified recently in China, predicting it's worldwide occurrence. In this study, a novel multilocus sequence typing (MLST) scheme was developed to analyse phylogenetic relationships between M. anserisalpingitidis field isolates and clinical specimens originating from different geographical locations. Five loci (atpG, fusA, pgiB, plsY, and uvrA) were selected for the final MLST study. The examined 89 M. anserisalpingitidis samples yielded 76 unique sequence types with a 0.994 Simpson's index of diversity. The samples were originated from Hungary, Poland, Ukraine, China, and Vietnam. Phylogenetic analysis revealed the existence of three distinct clades (A-C) and six subclades within clade C. Generally, samples originating from the same geographical locations or livestock integration clustered together. Isolates in clade A showed the closest relationships to the M. anatis outgroup due to sequence similarity of the plsY locus. The highest genetic distance was observed in 5C among the subclades of clade C, containing the Asian and some Hungarian field isolates. The developed MLST assay revealed high diversity of the investigated M. anserisalpingitidis samples. The method proved to be a valuable and cost-effective tool for sequence typing of this waterfowl Mycoplasma species, enabling the better understanding of its phylogeny and providing a robust assay for future molecular epidemiological investigations.

A new scheme for strain typing of methicillin-resistant Staphylococcus aureus on the basis of matrix-assisted laser desorption ionization time-of-flight mass spectrometry by using machine learning approach.

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most important clinical pathogens, conducts an increasing number of morbidity and mortality in the world. Rapid and accurate strain typing of bacteria would facilitate epidemiological investigation and infection control in near real time. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and cost-effective tool for presumptive strain typing. To develop robust method for strain typing based on MALDI-TOF spectrum, machine learning (ML) is a promising algorithm for the construction of predictive model. In this study, a strategy of building templates of specific types was used to facilitate generating predictive models of methicillin-resistant Staphylococcus aureus (MRSA) strain typing through various ML methods. The strain types of the isolates were determined through multilocus sequence typing (MLST). The area under the receiver operating characteristic curve (AUC) and the predictive accuracy of the models were compared. ST5, ST59, and ST239 were the major MLST types, and ST45 was the minor type. For binary classification, the AUC values of various ML methods ranged from 0.76 to 0.99 for ST5, ST59, and ST239 types. In multiclass classification, the predictive accuracy of all generated models was more than 0.83. This study has demonstrated that ML methods can serve as a cost-effective and promising tool that provides preliminary strain typing information about major MRSA lineages on the basis of MALDI-TOF spectra.

A new cost and time effective method for multilocus microsatellite typing (MLMT) studies: Application of Leishmania tropica isolates and clinical samples from Turkey.

Molecular techniques are widely used in the field of parasitology to identify the genetic profile of the microbiological agents. Microsatellite typing studies are comprised of the amplification of polymorphic markers to analyze the fragment sizes using bioinformatics tools. Current methods need fluorescently labeled primers and size markers to obtain fragment peaks in ABI PRISM® systems and due to low discrimination power of gel-electrophoresis, it is not possible to differentiate primer-dimers from small fragments In the present study, we designed a new method for fragment analysis studies, which reduce the time by eliminating the classical PCR, the gel-electrophoresis and the preparation steps of fragment analysis. Ten previously studied Leishmania tropica strains and one Giemsa-stained slide were tested by new method and obtained fragment peaks were compared to the previous data obtained from ABI PRISM® system. Overall twelve makers were tested and the signal peak from each fragment was compared to classical ABI PRISM®-based fragment analysis and noted as identical. The new protocol is time saving, cost effective, and eliminates the human error comparing to classical MLMT analysis protocol. We believe that this method enables the easy detection of the fragment lengths without having bioinformatics experience and the obtained data can be easily shared with other laboratories.

Next-generation sequencing for high-throughput molecular ecology: a step-by-step protocol for targeted multilocus genotyping by pyrosequencing.

Next-generation sequencing technology can now provide population biologists and phylogeographers with information at the genomic scale; however, many pertinent questions in population genetics and phylogeography can be answered effectively with modest levels of genomic information. For the past two decades, most population-level studies have lacked nuclear DNA (nDNA) sequence data due to the complications and cost of amplifying and sequencing diploid loci. However, pyrosequencing of emulsion PCR reactions, amplifying from only one molecule at a time, can generate megabases of clonally amplified loci at high coverage, thereby greatly simplifying allelic sequence determination. Here, we present a step-by-step methodology for utilizing the 454 GS FLX Titanium pyrosequencing platform to simultaneously sequence 16 populations (at 20 individuals per population) at 10 different nDNA loci (3,200 loci in total) in one plate of sequencing for less than the cost of traditional Sanger sequencing.

Comparison of multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) fingerprinting for differentiation of Campylobacter jejuni isolated from broiler in Chiang Mai, Thailand.

We compared rapid fingerprinting using repetitive sequence-based PCR (rep-PCR) for subtyping Campylobacter jejuni isolates to the widely used multilocus sequence typing (MLST). Representative C. jejuni isolates (n = 16) from broilers were analyzed using MLST and rep-PCR. Both techniques demonstrated an equal discriminatory power of 0.8917, and 9 subgroups were identified. Clonal identification of all 16 isolates was identical for both techniques. The rep-PCR as described in this study may be used as a rapid and cost-effective alternative for subtyping of C. jejuni isolates, or as an effective screening tool in large epidemiological studies.

High-resolution melting system to perform multilocus sequence typing of Campylobacter jejuni.

Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.

Analysis of multilocus sequence typing schemes for 35 different bacteria revealed that gene loci of 10 bacteria could be replaced to improve cost-effectiveness.

Although multilocus sequence typing (MLST) has been widely used for bacterial typing, the contribution of the gene loci to the discriminatory power of each MLST scheme is unknown. We analyzed the discriminatory powers of 36 MLST schemes using all combinations of the 7 loci and contributions of each locus to the schemes. In 10 schemes, sequencing 6 loci can achieve the discriminatory powers of 7 loci. For the other 26 schemes, the median marginal increase in discriminatory power when 7 instead of 6 loci were used is 0.0004. Sequencing the 7 loci of 50 strains each of Pseudomonas aeruginosa and Acinetobacter baumannii revealed that the discriminatory power for P. aeruginosa was 0.9861 when either 6 (without trp) or 7 loci were used and that for A. baumannii was 0.9363 when 5, 6, or 7 loci were used. Genes that have no additional or minimal contribution to the overall discriminatory powers should be replaced.