Conventional Multilocus Sequence Typing Research Articles

Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson’s ID (0.992) than conventional MLST (Simpson’s ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.

Antimicrobial resistance characteristics and phylogenetic relationships of pleuromutilin-resistant Enterococcus isolates from different environmental samples along a laying hen production chain

Antimicrobial resistance in the laying hen production industry has become a serious public health problem. The antimicrobial resistance and phylogenetic relationships of the common conditional pathogen Enterococcus along the laying hen production chain have not been systematically clarified. 105 Enterococcus isolates were obtained from 115 environmental samples (air, dust, feces, flies, sewage, and soil) collected along the laying hen production chain (breeding chicken, chick, young chicken, and commercial laying hen). These Enterococcus isolates exhibited resistance to some clinically relevant antibiotics, such as tetracycline (92.4%), streptomycin (92.4%), and erythromycin (91.4%), and all strains had multidrug resistance phenotypes. Whole genome sequencing characterized 29 acquired antibiotic resistance genes (ARGs) that conferred resistance to 11 classes of antibiotics in 51 pleuromutilin-resistant Enterococcus isolates, and lsa(E), which mediates resistance to pleuromutilins, always co-occurred with lnu(B). Alignments with the Mobile Genetic Elements database identified four transposons (Tn554, Tn558, Tn6261, and Tn6674) with several ARGs (erm(A), ant(9)-la, fex(A), and optrA) that mediated resistance to many clinically important antibiotics. Moreover, we identified two new transposons that carried ARGs in the Tn554 family designated as Tn7508 and Tn7492. A complementary approach based on conventional multi-locus sequence typing and whole genome single nucleotide polymorphism analysis showed that phylogenetically related pleuromutilin-resistant Enterococcus isolates were widely distributed in various environments on different production farms. Our results indicate that environmental contamination by antimicrobial-resistant Enterococcus requires greater attention, and they highlight the risk of pleuromutilin-resistant Enterococcus and ARGs disseminating along the laying hen production chain, thereby warranting effective disinfection.

Occurrence, antimicrobial resistance and whole genome sequence analysis of Salmonella serovars from pig farms in Ilorin, North-central Nigeria

Salmonella enterica is a foodborne pathogen of global public health importance with developing countries mostly affected. Foodborne outbreaks are often attributed to pork consumption and Salmonella contamination of retail pork is directly linked to the Salmonella prevalence on farm.The widespread use of antimicrobials at different steps of swine production can favor resistant strains of Salmonella. The objectives of this study are to characterize the distribution, multilocus sequence typing (MLST), plasmid, virulence profiles and antimicrobial resistance of Salmonella serovars circulating in selected pig farms.Six hundred fecal samples were randomly collected from nine selected farms in Ilorin, Nigeria. Isolates were analyzed by cultural isolation using selective media, conventional biochemical characterization, serotyping, MLST and whole genome sequencing (WGS).Sixteen samples were positive for Salmonella sub-species, comprising of nine serovars. The antimicrobial susceptibility results revealed low-level resistance against 13 antimicrobial agents. Five strains exhibited resistance to nalidixic acid and intermediate resistance to ciprofloxacin with chromosomal (double) mutation at gyrA and parC while four strains possessed single mutation in parC. Salmonella Kentucky showed double mutation each at gyrA and parC. WGS analysis, revealed eight diverse sequence types (STs), the most common STs were ST-321 and ST-19 (n = 4) exhibited by S. Muenster and S. Typhimurium, respectively. Single Nucleotide Polymorphism (SNP)-based phylogeny analysis showed the 16 isolates to be highly related and fell into 8 existing clusters at NCBI Pathogen Detection.Curtailing the spread of resistant strains will require the establishment of continuous surveillance program at the state and national levels in Nigeria. This study provides useful information for further studies on antimicrobial resistance mechanisms in foodborne Salmonella species.

Culture-independent multilocus sequence typing of Pseudomonas aeruginosa for cross-infection screening

The genotyping of pathogens within cystic fibrosis cohorts is an important process, enabling the detection of transmissible and clinically-important strains. Traditionally this has been via culture-dependent processes. However, culture-independent investigation of respiratory samples is becoming more common, with such approaches highlighting the limitations of culture-based methods. In this study we describe the culture-independent application of multilocus sequence typing (MLST) for Pseudomonas aeruginosa, performed on DNA extracted from the sputa of cystic fibrosis patients. We compare the output to conventional culture-dependent MLST applied to the same samples and demonstrate high concordance. Culture-independent MLST enabled genotyping of culture-negative samples in patients from whom P. aeruginosa was intermittently isolated, and revealed the hidden presence of transmissible strains. Culture-independent MLST is also capable of highlighting samples containing multiple strains, albeit inconsistently. We conclude that culture-independent MLST can be a useful genotyping tool for screening cohorts and identifying patients that warrant further detailed investigation.

Emergence of vancomycin-resistant Enterococcus faecium ST1421 lacking the pstS gene in Korea.

Although multilocus sequence typing (MLST) has been used to study molecular epidemiology and to explore the population structure of Enterococcus faecium, vancomycin-resistant E. faecium (VREF) strains lacking the pstS gene that were non-typable using conventional MLST methods were reported recently. We found nationwide emergence of VREF isolates lacking pstS in Korea and hereby report the molecular characteristics of these isolates. Forty-six VREF isolates lacking the pstS gene were identified among 300 VREF rectal isolates collected from hospitalized patients between 2014 and 2015. MLST was performed and clonal relatedness was determined by pulsed-field gel electrophoresis (PFGE). Four VREF ST1421 isolates were whole-genome sequenced. Among the VREF rectal isolates lacking pstS, 98% were classified as ST1421, which has identical allelic profiles to ST17 for all housekeeping genes except pstS. PFGE pattern analyses revealed 32 pulsotypes. All isolates harbored Tn1546 components with various transposase and insertion sequences. The whole-genome sequencing of four VREF ST1421 isolates showed that the pstS gene region was deleted at various locations with considerable inversion. The pstS gene was also depleted in 12.1% of 33 VREF clinical isolates in 2006-2007 and in 11.8% of 59 clinical isolates in 2012-2013. VREF ST1421 strains lacking the pstS gene have emerged in Korea. The emergence and spread of pstS-deleted VREF strains pose a serious challenge for epidemiological investigation. Alternative molecular typing methods to MLST will be increasingly necessary.

The Complexity of the Identification of Burkholderia cepacia Strain Which Caused Septicemia

Background: The correct identifying of pathogenic Burkholderia spp. using available commercial biochemical systems is a certain problem due to metabolic plasticity and variable enzymatic profile of isolates. Objectives: The current study aimed at using specific PCR and conventional multi-locus sequence typing (MLST) scheme to confirm the uncertain identification results for Burkholderia cepacia clinical isolate. Methods: Multilocus species-specific PCR and MLST profiling of high-throughput sequencing data have been used to clarify the varied results of biochemical identification of the strain. Results: The strain isolated from a patient with septicemia was initially identified as B. pseudomallei by Vitek 2 GN system but has an uncharacteristic antimicrobial resistance pattern and colony morphology. A species-specific multilocus PCR and whole-genome sequence profiling, according to the MLST scheme, allowed to identify an isolate as B. cepacia. Conclusions: The obtained results demonstrate the preference of molecular tests for correct identifying of pathogenic Burkholderia, considering that misidentification of those may lead to improper treatment or increase of biosafety risk.

Long-Term, Low-Frequency Cluster of a German-Imipenemase-1-Producing Enterobacter hormaechei ssp. steigerwaltii ST89 in a Tertiary Care Hospital in Germany.

Enterobacter cloacae complex is a common cause of hospital outbreaks. A retrospective and prospective molecular analysis of carbapenem-resistant clinical isolates in a tertiary care center demonstrated an outbreak of a German-imipenemase-1 (GIM-1) metallo-beta-lactamase-producing Enterobacter hormaechei ssp. steigerwaltii affecting 23 patients between 2009 and 2016. Thirty-three isolates were sequence type 89 by conventional multilocus sequence typing (MLST) and displayed a maximum difference of 49 out of 3,643 targets in the ad-hoc core-genome MLST (cgMLST) scheme (SeqSphere+ software; Ridom, Münster, Germany). The relatedness of all isolates was confirmed by further maximum-likelihood phylogeny. One clonal complex of highly related isolates (≤15 allele difference in cgMLST) contained 17 patients, but epidemiological data only suggested five transmission events. The blaGIM-1-gene was embedded in a class-1-integron (In770) and the Tn21-subgroup transposon Tn6216 (KC511628) on a 25-kb plasmid. Environmental screening detected one colonized sink trap in a service room. The outbreak was self-limited as no further blaGIM-1-positive E. hormaechei has been isolated since 2016. Routine molecular screening of carbapenem-nonsusceptible gram-negative isolates detected a long-term, low-frequency outbreak of a GIM-1-producing E. hormaechei ssp. steigerwaltii clone. This highlights the necessity of molecular surveillance.

Using whole genome sequencing to study American foulbrood epidemiology in honeybees.

American foulbrood (AFB), caused by Paenibacillus larvae, is a devastating disease in honeybees. In most countries, the disease is controlled through compulsory burning of symptomatic colonies causing major economic losses in apiculture. The pathogen is endemic to honeybees world-wide and is readily transmitted via the movement of hive equipment or bees. Molecular epidemiology of AFB currently largely relies on placing isolates in one of four ERIC-genotypes. However, a more powerful alternative is multi-locus sequence typing (MLST) using whole-genome sequencing (WGS), which allows for high-resolution studies of disease outbreaks. To evaluate WGS as a tool for AFB-epidemiology, we applied core genome MLST (cgMLST) on isolates from a recent outbreak of AFB in Sweden. The high resolution of the cgMLST allowed different bacterial clones involved in the disease outbreak to be identified and to trace the source of infection. The source was found to be a beekeeper who had sold bees to two other beekeepers, proving the epidemiological link between them. No such conclusion could have been made using conventional MLST or ERIC-typing. This is the first time that WGS has been used to study the epidemiology of AFB. The results show that the technique is very powerful for high-resolution tracing of AFB-outbreaks.

A rapid typing method for Listeria monocytogenes based on high-throughput multilocus sequence typing (Hi-MLST)

Listeria monocytogenes infects humans via food products, causing listeriosis. Consequently, food companies pay meticulous attention to the risk of contamination of their products by this bacterium. While fragment analysis methods such as pulsed-field gel electrophoresis (PFGE) are used to trace the sources of contamination for this bacterium, some drawbacks have been identified, namely the complexity of the methods and the difficulty of making data comparisons. As an alternative, multilocus sequence typing (MLST) is now seeing widespread use; however, owing to its cost, time, and labor requirements, its diffusion into the food industry has been slow. Thus, in the present study, a High-throughput MLST (Hi-MLST) method, which can rapidly, simply, and cheaply perform MLST analyses using a next-generation sequencer (NGS) that can analyze a large volume of base sequences at once was developed. Firstly, a multiplex PCR method designed to amplify seven genes for use in MLST was developed. The discriminatory potential of the developed method was confirmed in silico, and was verified that it has the same discriminatory potential as conventional methods. Next, MLST analysis using multiplex PCR and NGS was performed for 48 strains of L. monocytogenes. The sequences obtained from this analysis have sufficiently reliable quality for all of the genes from of all the strains. Thus, this method could classify the 48 strains into 39 sequence types (ST) with a Diversity index (DI) of 0.989. In summary, using the Hi-MLST method developed in the present study, which combined multiplex PCR and NGS, cut the costs to 1/6th and the time to 1/20th that of conventional MLST methods.

Efficient, Cost-Effective, High-Throughput, Multilocus Sequencing Typing (MLST) Method, NGMLST, and the Analytical Software Program MLSTEZ.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species. It can be used to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. However, conventional MLST is costly and time consuming, which limits its power for genotyping large numbers of samples. 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 a single assay.

MOST: a modified MLST typing tool based on short read sequencing.

Multilocus sequence typing (MLST) is an effective method to describe bacterial populations. Conventionally, MLST involves Polymerase Chain Reaction (PCR) amplification of housekeeping genes followed by Sanger DNA sequencing. Public Health England (PHE) is in the process of replacing the conventional MLST methodology with a method based on short read sequence data derived from Whole Genome Sequencing (WGS). This paper reports the comparison of the reliability of MLST results derived from WGS data, comparing mapping and assembly-based approaches to conventional methods using 323 bacterial genomes of diverse species. The sensitivity of the two WGS based methods were further investigated with 26 mixed and 29 low coverage genomic data sets from Salmonella enteridis and Streptococcus pneumoniae. Of the 323 samples, 92.9% (n = 300), 97.5% (n = 315) and 99.7% (n = 322) full MLST profiles were derived by the conventional method, assembly- and mapping-based approaches, respectively. The concordance between samples that were typed by conventional (92.9%) and both WGS methods was 100%. From the 55 mixed and low coverage genomes, 89.1% (n = 49) and 67.3% (n = 37) full MLST profiles were derived from the mapping and assembly based approaches, respectively. In conclusion, deriving MLST from WGS data is more sensitive than the conventional method. When comparing WGS based methods, the mapping based approach was the most sensitive. In addition, the mapping based approach described here derives quality metrics, which are difficult to determine quantitatively using conventional and WGS-assembly based approaches.

Global MLST of Salmonella Typhi Revisited in Post-genomic Era: Genetic Conservation, Population Structure, and Comparative Genomics of Rare Sequence Types.

Typhoid fever, caused by Salmonella enterica serovar Typhi, remains an important public health burden in Southeast Asia and other endemic countries. Various genotyping methods have been applied to study the genetic variations of this human-restricted pathogen. Multilocus sequence typing (MLST) is one of the widely accepted methods, and recently, there is a growing interest in the re-application of MLST in the post-genomic era. In this study, we provide the global MLST distribution of S. Typhi utilizing both publicly available 1,826 S. Typhi genome sequences in addition to performing conventional MLST on S. Typhi strains isolated from various endemic regions spanning over a century. Our global MLST analysis confirms the predominance of two sequence types (ST1 and ST2) co-existing in the endemic regions. Interestingly, S. Typhi strains with ST8 are currently confined within the African continent. Comparative genomic analyses of ST8 and other rare STs with genomes of ST1/ST2 revealed unique mutations in important virulence genes such as flhB, sipC, and tviD that may explain the variations that differentiate between seemingly successful (widespread) and unsuccessful (poor dissemination) S. Typhi populations. Large scale whole-genome phylogeny demonstrated evidence of phylogeographical structuring and showed that ST8 may have diverged from the earlier ancestral population of ST1 and ST2, which later lost some of its fitness advantages, leading to poor worldwide dissemination. In response to the unprecedented increase in genomic data, this study demonstrates and highlights the utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure.

Core Genome Multilocus Sequence Typing Scheme for High- Resolution Typing of Enterococcus faecium.

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism(SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates.

Multilocus sequence typing of Australian Streptococcus suis type 2 by MALDI-TOF mass spectrometry analysis of PCR amplicons

Streptococcus suis serotype 2 is a ubiquitous pathogen of swine and is known to cause severe disease in humans. Multilocus sequence typing (MLST) is ideal for characterising this organism because it permits isolates to be compared on a national and international scale. A novel approach to MLST using matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MS-MLST) provides a more rapid alternative to dideoxy sequencing. This study used MS-MLST to define the multilocus sequence types (STs) present among a collection of Australian S. suis type 2, and thus, delivered a basis for comparison of Australian isolates with international strains already well characterised for virulence attributes. A collection of 45 isolates recovered from infected humans (n=3) and diseased pigs (n=42) was genotyped using MS-MLST and conventional MLST. Both methods were 100% concordant in their classification of sequence types (STs), although MS-MLST permitted much quicker analysis of sequence data. The collection contained ST25 (n=31), ST1 (n=10), ST28 (n=3) and ST369 (n=1). These results are consistent with the population structure of S. suis type 2 observed in diseased pigs and humans in Canada and the United Kingdom. MS-MLST may have utility for studying the population structure and epidemiology of S. suis in countries where the diversity of S. suis is greater and human disease is more common.

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.

P4-S1.06 High-resolution Multilocus Sequence Typing (MLST) of Chlamydia trachomatis from three catchment areas in North and Central Norway

BackgroundThe C trachomatis incidence rate in the county of Finnmark in North Norway is twice as high as in the rest of the country. This study genetically characterises the C...

High-Resolution Genotyping of Campylobacter Species by Use of PCR and High-Throughput Mass Spectrometry

In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which "weighs" PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.