Within patient genetic diversity of blaKPC harboring Klebsiella pneumoniae in a Colombian hospital and identification of a new NTEKPC platform.

Resistance to carbapenems in Klebsiella pneumoniae has been mostly related with the worldwide dissemination of KPC, largely due to the pandemic clones belonging to the complex clonal (CC) 258. To unravel blaKPC post-endemic clinical impact, here we describe clinical characteristics of 68 patients from a high complexity hospital, and the molecular and genetic characteristics of their 139 blaKPC-K. pneumoniae (KPC-Kp) isolates. Of the 26 patients that presented relapses or reinfections, 16 had changes in the resistance profiles of the isolates recovered from the recurrent episodes. In respect to the genetic diversity of KPC-Kp isolates, PFGE revealed 45 different clonal complexes (CC). MLST for 12 representative clones showed ST258 was present in the most frequent CC (23.0%), however, remaining 11 representative clones belonged to non-CC258 STs (77.0%). Interestingly, 16 patients presented within-patient genetic diversity of KPC-Kp clones. In one of these, three unrelated KPC-Kp clones (ST258, ST504, and ST846) and a blaKPC-K. variicola isolate (ST182) were identified. For this patient, complete genome sequence of one representative isolate of each clone was determined. In K. pneumoniae isolates blaKPC was mobilized by two Tn3-like unrelated platforms: Tn4401b (ST258) and Tn6454 (ST504 and ST846), a new NTEKPC-IIe transposon for first time characterized also determined in the K. variicola isolate of this study. Genome analysis showed these transposons were harbored in different unrelated but previously reported plasmids and in the chromosome of a K. pneumoniae (for Tn4401b). In conclusion, in the blaKPC post-endemic dissemination in Colombia, different KPC-Kp clones (mostly non-CC258) have emerged due to integration of the single blaKPC gene in new genetic platforms. This work also shows the intra-patient resistant and genetic diversity of KPC-Kp isolates. This circulation dynamic could impact the effectiveness of long-term treatments.

Genetic diversity of Francisella tularensis in Poland with comments on MLVA genotyping and a proposition of a novel rapid v4-genotyping.

We investigated the genotypes of Francisella tularensis (F. tularensis) strains isolated in Poland during the period 1953-2013 and studied their genetic relationship to F. tularensis strains isolated in other countries using MLVA. We examined the mosquito and tick samples collected in Poland for the presence of F. tularensis DNA using PCR. Our results revealed a high genetic diversity among the strains of F. tularensis collected from Poland, suggesting that the bacterium is commonly found in the environment. However, we did not detect F. tularensis DNA in ticks and mosquitoes, showing that the arthropod bites might not be the main source of infection. We also propose the application of a practical assay called v4-genotyping that can be directly performed on the clinical and environmental samples. In addition, we discovered genetic variations among Schu S4 reference strains used in various laboratories and showed that MLVA analysis should not be based on amplicon sizes only because point mutations occurring within the MLVA loci might not always be manifested by a change in the amplicon size.

MentaLiST - A fast MLST caller for large MLST schemes.

MLST (multi-locus sequence typing) is a classic technique for genotyping bacteria, widely applied for pathogen outbreak surveillance. Traditionally, MLST is based on identifying sequence types from a small number of housekeeping genes. With the increasing availability of whole-genome sequencing data, MLST methods have evolved towards larger typing schemes, based on a few hundred genes [core genome MLST (cgMLST)] to a few thousand genes [whole genome MLST (wgMLST)]. Such large-scale MLST schemes have been shown to provide a finer resolution and are increasingly used in various contexts such as hospital outbreaks or foodborne pathogen outbreaks. This methodological shift raises new computational challenges, especially given the large size of the schemes involved. Very few available MLST callers are currently capable of dealing with large MLST schemes. We introduce MentaLiST, a new MLST caller, based on a k-mer voting algorithm and written in the Julia language, specifically designed and implemented to handle large typing schemes. We test it on real and simulated data to show that MentaLiST is faster than any other available MLST caller while providing the same or better accuracy, and is capable of dealing with MLST schemes with up to thousands of genes while requiring limited computational resources. MentaLiST source code and easy installation instructions using a Conda package are available at https://github.com/WGS-TB/MentaLiST.

Microbial sequence typing in the genomic era.

Next-generation sequencing (NGS), also known as high-throughput sequencing, is changing the field of microbial genomics research. NGS allows for a more comprehensive analysis of the diversity, structure and composition of microbial genes and genomes compared to the traditional automated Sanger capillary sequencing at a lower cost. NGS strategies have expanded the versatility of standard and widely used typing approaches based on nucleotide variation in several hundred DNA sequences and a few gene fragments (MLST, MLVA, rMLST and cgMLST). NGS can now accommodate variation in thousands or millions of sequences from selected amplicons to full genomes (WGS, NGMLST and HiMLST). To extract signals from high-dimensional NGS data and make valid statistical inferences, novel analytic and statistical techniques are needed. In this review, we describe standard and new approaches for microbial sequence typing at gene and genome levels and guidelines for subsequent analysis, including methods and computational frameworks. We also present several applications of these approaches to some disciplines, namely genotyping, phylogenetics and molecular epidemiology.

The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

Multiple-Locus Variable Number Tandem Repeat Analysis of Klebsiella pneumoniae: Comparison with Pulsed-Field Gel Electrophoresis.

AIM: To assess whether multiple-locus variable-number tandem repeat analysis (MLVA) could replace pulsed-field gel electrophoresis (PFGE) for genotyping of Klebsiella pneumoniae, this study was conducted to compare the typeability, discriminatory power, and concordance of these methods. MATERIALS AND METHODS: We used the nine variable number tandem repeat (VNTR) loci scheme to test its suitability for differentiating 114 ESBL-producing K. pneumoniae isolates collected from different clinical specimens in three hospitals of Tehran, Iran between April and December 2011. PFGE with XbaI was performed and the results were compared with those obtained by typing with MLVA. RESULTS: MLVA and PFGE yielded 44 and 64 types, respectively. Simpson's Diversity Index of MLVA was 0.896 (a 95% confidence interval of 0.850-0.942) and of PFGE was 0.962 (a 95% confidence interval of 0.943-0.981). Congruence between PFGE and MLVA was low. The adjusted Wallace coefficient of PFGE to MLVA was 0.946; however, MLVA was less able to predict PFGE type (32.5%). A range of 2-12 alleles was identified at VNTR loci with Simpson's diversity values between 0.017 and 0.818. CONCLUSION: MLVA is a PCR-based typing method and is much easier and more rapid in comparison to PFGE. These data indicate that the MLVA typing scheme used in this study is discriminative and reliable for typing of K. pneumoniae. However, optimization of the VNTR markers is required to improve the discriminatory power of the method.

New lipid-dependent Malassezia species from parrots / Nuevas especies lipodependientes del género Malassezia procedentes de loros

Background. All the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals. Aim. In this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov. Methods. The isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed. Results. The strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species. Conclusions. These studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots (AU)

Antecedentes. Todas las especies del género Malassezia actualmente identificadas se han aislado de mamíferos. Sin embargo, tan solo unas pocas de ellas se han aislado de aves. De hecho, las aves han sido estudiadas con menos frecuencia como portadoras de estas levaduras que los mamíferos. Objetivos. En este estudio describimos dos nuevas especies del género Malassezia: Malassezia brasiliensis sp. nov. y Malassezia psittaci sp. nov. Métodos. Las cepas estudiadas en esta publicación se aislaron de loros utilizados como animales de compañía en Brasil. Las cepas se caracterizaron mediante los criterios morfológicos y fisiológicos actualmente utilizados para la identificación de estas levaduras. También se llevó a cabo la secuenciación y el análisis de los fragmentos génicos D1/D2 26S e ITS-5.8S del ADN ribosómico y del gen de la β-tubulina. Resultados. Los perfiles fenotípicos de las cepas propuestas como nuevas especies no encajaron completamente con los de las especies descritas en este género. Además, el análisis de los genes estudiados respaldó la validez de las nuevas especies. El análisis multilocus de secuencias de los tres loci estudiados reforzó con mayor firmeza la definición de las nuevas especies. Conclusiones. Todos estos estudios confirman la separación de estas dos nuevas especies del resto de las especies descritas del género Malassezia, así como la existencia de especies dependientes de lípidos del género Malassezia en loros (AU)

Development and application of MLVA methods as a tool for inter-laboratory surveillance.

Multiple-locus variable-number of tandem-repeats analysis (MLVA) has emerged as a valuable method for subtyping bacterial pathogens and has been adopted in many countries as a critical component of their laboratory-based surveillance. Lack of harmonisation and standardisation of the method, however, has made comparison of results generated in different laboratories difficult, if not impossible, and has therefore hampered its use in international surveillance. This paper proposes an international consensus on the development, validation, nomenclature and quality control for MLVA used for molecular surveillance and outbreak detection based on a review of the current state of knowledge.

Proof-of-concept study for successful inter-laboratory comparison of MLVA results.

Multiple-locus variable-number of tandem repeats analysis (MLVA) is widely used for typing of pathogens. Methods such as MLVA based on determining DNA fragment size by the use of capillary electrophoresis have an inherent problem as a considerable offset between measured and real (sequenced) lengths is commonly observed. This discrepancy arises from variation within the laboratory set-up used for fragment analysis. To obtain comparable results between laboratories using different set-ups, some form of calibration is a necessity. A simple approach is to use a set of calibration strains with known allele sizes and determine what compensation factors need to be applied under the chosen set-up conditions in order to obtain the correct allele sizes. We present here a proof-of-concept study showing that using such a set of calibration strains makes inter-laboratory comparison possible. In this study, 20 international laboratories analysed 15 test strains using a five-locus Salmonella enterica serovar Typhimurium MLVA scheme. When using compensation factors derived from a calibration set of 33 isolates, 99.4% (1,461/1,470) of the MLVA alleles of the test strains were assigned correctly, compared with 64.8% (952/1,470) without any compensation. After final analysis, 97.3% (286/294) of the test strains were assigned correct MLVA profiles. We therefore recommend this concept for obtaining comparable MLVA results.

Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.

Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.