Sequence Typing Scheme Research Articles

Zoonotic Giardia duodenalis assemblage A in northern sloth from Brazilian Amazon.

The parasite Giardia duodenalis infects a wide range of vertebrate hosts, including domestic and wild animals as well as humans. Giardia is genotyped into eight assemblages (A-H). Zoonotic assemblages A and B have already been identified in humans and wild and domestic animals (non-human primates and cats) from Brazilian Amazon and in the world. Due to its zoonotic/zooanthroponotic nature, surveillance initiatives and the definition of Giardia assemblages are important in order to characterise the epidemiological scenario and to implement further control measures. Determine assemblages of G. duodenalis in sloths from the Brazilian Amazon Region. Faecal parasitological examination of sloths from Amazonas State. Polymerase chain reaction (PCR) targeting the beta giardin (BG), and genes from multilocus sequence typing (MLST) scheme, amplicon sequencing and phylogenetic analysis. Here, we identified, by microscopy, Giardia in two northern sloths (Bradypus tridactylus). These two samples were submitted to molecular assays and it was revealed that both were infected by G. duodenalis assemblage A. Phylogenetic analysis showed that they belong to assemblage A within sequences from humans and wild and domestic animals. Therefore, besides showing, by the first time, the current presence of this parasite in sloths, our findings reveals that this wild animal species would be part of the zoonotic/zooanthroponotic scenario of this parasite in the Brazilian Amazon.

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power

BackgroundThe flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers.MethodsWe genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates.ResultsWe identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates.ConclusionThe MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.Graphical

Utility evaluation of two molecular methods for Leptospira spp. typing in human serum samples

Most of the available genotyping methods were applied and evaluated in Leptospira isolates and only few of them in a relevant sample size of blood specimens but not of sera. The objective of this study was to evaluate the utility of one partial 16S rRNA gene sequencing assay (16S rRNA) and an optimized. Multilocus sequence typing scheme (MLST) for Leptospira typing directly in serum samples. Confirmed leptospirosis patients (n = 228) from Argentina (2005–2016) were randomly included. Septicemic-phase serum samples (n = 228) were studied by two genotyping methods. Available immune-phase serum samples of the included patients (n = 159) were studied by MAT to compare serological and molecular results. In culture-proven cases (n = 8), genotyping results between clinical samples and isolates were compared. Typing success rate (TSR) was 21.9% for 16S rRNA and 11.4% for MLST (full allelic profile) and a positive trend in both TSR during the study period was observed. Two species (L. interrogans and L. borgpertesenii) were identified by both methods and MLST assigned 8 different STs. The probable serogroups identified by MLST were coincident with the presumptive infecting serogroups identified by MAT, but with different frequencies. The three serogroups (Canicola, Sejroe and Icterohaemorrhagiae) most frequently identified by MAT were also genotyped by MLST. Typing results via 16S rRNA and MLST in clinical samples and isolates of culture-proven cases, were consistent except for one case. Performance of partial 16S rRNA gene sequencing assay and the optimized MLST scheme directly in sera may increase and improve the knowledge about species and serogroups causing human leptospirosis, especially in countries with low rates of culture sample collection or Leptospira isolation.

Specific high-resolution scheme to improve understanding of the spatio-temporal dispersion of lymphogranuloma venereum epidemic.

Lymphogranuloma venereum (LGV) is already endemic in vulnerable populations in several European countries; however, molecular epidemiology data with improved accuracy are necessary to better understand LGV epidemic in these countries. Current strategies to study the molecular epidemiology of LGV cases involve schemes based on a few genetic fragments of Chlamydia trachomatis, which have demonstrated limited discriminatory power for LGV. Therefore, this study aimed to propose a new combination of molecular markers based on the most variable genes of L-genotype genomes to improve the characterization of the current LGV epidemic in Madrid, Spain. Four genes were selected according to their diversity index (CTLon_0054, CTLon_0087, CTLon_0243 and CTLon_0301) for use in combination with ompA. In silico and experimental studies were performed to compare the previously described multilocus sequence typing (MLST) schemes with our proposal. Moreover, the proposed scheme was applied (n = 68) to analyze the spatio-temporal spread of the LGV cases. Our proposal demonstrated higher diversity allowing the identification of three main groups compared to the previously published MLST based on hypervariable genes wherein only a single sequence type was identified. The temporal analysis showed that the major cluster was progressively diversifying, revealing a very active transmission chain. Furthermore, an L2b genome identical to that of the origin of the epidemic was detected, suggesting reintroductions or a low screening rate in vulnerable populations. The spatial distribution suggests that the selection and spread of new variants occurs from the central district to the peripheral regions. The scheme proposed in this study has proven to be useful for appropriate discrimination of LGV strains. This study, to our knowledge for the first time, demonstrates a spatio-temporal spread that increases our understanding and identifies areas with special susceptibility for maintenance of the endemic situation of LGV.

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis Assemblage A confirms its good discriminatory power

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis Assemblage A confirms its good discriminatory power

Molecular Genetic Epidemiology of an Emerging Antimicrobial-Resistant Klebsiella pneumoniae Clone (ST307) Obtained from Clinical Isolates in Central Panama.

We conducted an epidemiological study in three reference hospitals in the central region of Panama, using isolates of K. pneumoniae involved in infections, and identifying their AMR profile, associated clinical risk factors, and molecular typing using a multilocus sequence typing (ST) scheme. Six STs were detected: 307 (55%), 152, 18, 29, 405, and 207. CTX-M-15- and TEM-type beta-lactamases were identified in 100% of ESBL-producing strains; substitutions in gyrA Ser83Ile and parC Ser80Ile were identified in all ST307s; and in ST152 gyrA Ser83Phe, Asp87Ala, and parC Ser80Ile, the qnrB gene was detected in all strains resistant to ciprofloxacin. We present the first report on ST307 in three reference hospitals in the central region of Panama, which is a high-risk emerging clone and represents a public health alert for potential difficulties in managing K. pneumoniae infections in Panama, and which may extend to other Central American countries.

CUTIS-SEQ, a flexible bilocus sequence typing scheme that provides high resolution of Cutibacterium acnes strains across all subspecies

ObjectivesA ‘high resolution’ Single Locus Sequence Typing (SLST) scheme has been described for the anaerobic skin bacterium Cutibacterium acnes that seemingly discriminates sequence types (STs) to a level commensurate with previously described Multilocus Sequence Typing (MLST) methods (MLST4; MLST8; MLST9). However, no quantifiable evaluation of SLST versus MLST for differentiation of C. acnes strains, especially in relation to the subspecies of the bacterium, known as C. acnes subsp. acnes (type I), C. acnes subsp. defendens (type II) and C. acnes subsp. elongatum (type III), has been performed which is vital given its increasing use. To address this, we examined the discriminatory power of SLST versus MLST with a large group of isolates representative of all subspecies. MethodsSimpson's index of diversity (D) was used for quantitative comparison of the resolving power of the SLST and MLST schemes for 186 isolates of C. acnes covering all three subspecies. ResultsWhen strains were considered collectively, SLST and all three MLST approaches had similar D values > 90%. However, at the subspecies level there were significant differences between the methods, most strikingly a reduced discrimination of type II and type III strains (D <80%) by SLST versus MLST8, and to a lesser extent MLST4. The MLST9 method also performed poorly for type II strains (D <70%), but did display the best results for type I (D = 90%). By combining the SLST locus with the camp2 gene sequence to create a novel and flexible high-resolution Bilocus Sequence Typing (BLST) scheme, known as CUTIS-SEQ typing (CUTIbacterium acneS BilocuS sEQuence Typing), we achieved improved resolution at both species and, critically, subspp. levels. ConclusionsCUTIS-SEQ provides an opportunity to improve differentiation of C. acnes isolates by SLST without significantly impacting laboratory workload, or compromising application to complex biological communities. A CUTIS-SEQ isolate database is now available as part of the C. acnes PubMLST database at https://pubmlst.org.

Comparative Pan-Genomic Analysis Revealed an Improved Multi-Locus Sequence Typing Scheme for Staphylococcus aureus.

The growing prevalence of antibiotic-resistant Staphylococcus aureus strains mandates selective susceptibility testing and epidemiological investigations. It also draws attention to an efficient typing strategy. Whole genome sequencing helps in genetic comparison, strain differentiation, and typing; however, it is not that cost-effective. In comparison, Multi-Locus Sequence Typing (MLST) is an efficient typing method employed for bacterial strain typing and characterizations. In this paper, a comprehensive pangenome and phylogenetic analysis of 502/1279 S. aureus genomes is carried out to understand the species divergence. Additionally, the current Multi-Locus Sequence Typing (MLST) scheme was evaluated, and genes were excluded or substituted by alternative genes based on reported shortcomings, genomic data, and statistical scores calculated. The data generated were helpful in devising a new Multi-Locus Sequence Typing (MLST) scheme for the efficient typing of S. aureus strains. The revised scheme is now a blend of previously used genes and new candidate genes. The genes yQil, aroE, and gmk are replaced with better gene candidates, opuCC, aspS, and rpiB, based on their genome localization, representation, and statistical scores. Therefore, the proposed Multi-Locus Sequence Typing (MLST) method offers a greater resolution with 58 sequence types (STs) in comparison to the prior scheme's 42 STs.

Tracing Foodborne Botulism Events Caused by Clostridium botulinum in Xinjiang Province, China, Using a Core Genome Sequence Typing Scheme.

Foodborne botulism is a rare but life-threatening illness resulting from the action of a potent toxin mainly produced by Clostridium botulinum. It grows in an oxygen-deficient environment and is extremely viable in meat and soy products, making it one of the most virulent bacteria. How to track foodborne botulism events quickly and accurately has become a key issue. Here, we investigated two foodborne botulism events that occurred in Xinjiang in 2019 based on whole-genome sequencing and also successfully traced the relationship between clinical and food C. botulinum isolates using whole-genome core gene markers. All 59 isolates were classified as group I strains. Of the strains isolated in this study, 44 were found to be botulinum toxin A(B), and 15 isolates contained only the toxin B locus. Both the toxin A and B gene segments were located on the chromosome and organized in an ha cluster. Antibiotic resistance and virulence factors were also investigated. A set of 329 universal core gene markers were established using C. botulinum strains from a public database. These core gene markers were applied to the published C. botulinum genomes, and three outbreaks were identified. This work demonstrates that universal core gene markers can be used to trace foodborne botulism events, and we hope that our work will facilitate this effort in future. IMPORTANCE In this study, we analyzed 59 foodborne botulism (FB)-related strains isolated in Xinjiang Province, China. Our findings not only reveal the group classification, neurotoxin locus organization, antibiotic resistance and virulence factors of these strains but also establish a set of core gene markers for tracing foodborne botulism events, which was verified using published genomes. These findings indicate that these gene markers might be used as a potential tracing tool for FB events caused by C. botulinum group I strains, which have relatively stable genomic components.

Characterisation of Staphylococci Isolated from Milk Samples of a Water Buffalo Herd.

Water buffalo produce a tenth of milk for global human consumption. Non-aureus staphylococci (NAS) are among the most commonly isolated bacteria from mastitis in water buffalo and dairy cows. These results described the initial characterisation of 17 NAS-15 Staphylococcus simulans and two Staphylococcus chromogenes from a water buffalo herd (n = 44) in South Africa. The isolates were identified by classical microbiology, MALDI-TOF, and 16S rRNA, and the disc diffusion method determined the antibiotic susceptibility. A multi-locus sequence typing scheme (MLST) was developed to determine S. simulans sequence types (ST), by defining and comparing seven housekeeping gene fragment sequences. Sequence typing confirmed all 15 S. simulans isolates from water buffalo which belonged to a single ST, genetically distant from the six bovine STs isolated from adjacent farms, which also varied, indicating no current bacterial transfer between species. The antibiotic resistance patterns of S. simulans varied between beta-lactams. The mean milk somatic cell count (SCC) for the water buffalo milk samples was 166,500 cells/mL milk. This information offers insights into the epidemiology and comparison among isolates from various origins, which leads to effective proactive mastitis strategies resulting in safe, high-quality dairy products from water buffalo and dairy cows for human consumption.

MGTdb: a web service and database for studying the global and local genomic epidemiology of bacterial pathogens.

Multilevel genome typing (MGT) enables the genomic characterization of bacterial isolates and the relationships among them. The MGT system describes an isolate using multiple multilocus sequence typing (MLST) schemes, referred to as levels. Thus, for a new isolate, sequence types (STs) assigned at multiple precisely defined levels can be used to type isolates at multiple resolutions. The MGT designation for isolates is stable, and the assignment is faster than the existing approaches. MGT's utility has been demonstrated in multiple species. This paper presents a publicly accessible web service called MGTdb, which enables the assignment of MGT STs to isolates, along with their storage, retrieval and analysis. The MGTdb web service enables upload of genome data as sequence reads or alleles, which are processed and assigned MGT identifiers. Additionally, any newly sequenced isolates deposited in the National Center for Biotechnology Information's Sequence Read Archive are also regularly retrieved (currently daily), processed, assigned MGT identifiers and made publicly available in MGTdb. Interactive visualization tools are presented to assist analysis, along with capabilities to download publicly available isolates and assignments for use with external software. MGTdb is currently available for Salmonella enterica serovars Typhimurium and Enteritidis and Vibrio cholerae. We demonstrate the usability of MGTdb through three case studies - to study the long-term national surveillance of S. Typhimurium, the local epidemiology and outbreaks of S. Typhimurium, and the global epidemiology of V. cholerae. Thus, MGTdb enables epidemiological and microbiological investigations at multiple levels of resolution for all publicly available isolates of these pathogens. Database URL: https://mgtdb.unsw.edu.au.

Ralstonia Strains from Potato-Growing Regions of Kenya Reveal Two Phylotypes and Epidemic Clonality of Phylotype II Sequevar 1 Strains.

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is the most destructive potato disease in Kenya. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with bacterial wilt of potato in Kenya, (ii) generate an RSSC distribution map for epidemiological inference, and (iii) determine whether phylotype II sequevar 1 strains exhibit epidemic clonality. Surveys were conducted in 2018 and 2019, in which tubers from wilting potato plants and stem samples of potential alternative hosts were collected for pathogen isolation. The pathogen was phylotyped by multiplex PCR and 536 RSSC strains typed at a sequevar level. Two RSSC phylotypes were identified, phylotype II (98.4%, n = 506 [sequevar 1 (n = 505) and sequevar 2 (n = 1)]) and phylotype I (1.6%, n = 30 [sequevar 13 (n = 9) and a new sequevar (n = 21)]). The phylotype II sequevar 1 strains were haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. The TRST scheme identified 51 TRST profiles within the phylotype II sequevar 1 strains with a modest diversity index (HGDI = 0.87), confirming the epidemic clonality of RSSC phylotype II sequevar 1 strains in Kenya. A minimum spanning tree and mapping of the TRST profiles revealed that TRST27 '8-5-12-7-5' is the primary founder of the clonal complex of RSSC phylotype II sequevar 1 and is widely distributed via latently infected seed tubers. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

S8.5c MLST genotyping and phylogenetics of AD-hybrids

S8.5 Genotyping of Cryptococcus neoformans and C. gattii, September 23, 2022, 3:00 PM - 4:30 PMObjectivesIn a previous study a set of new molecular-type specific primers were designed to apply the standard ISHAM consensus multi-locus sequence typing (MLST) scheme to Cryptococcus neoformans AD hybrids. In the present study, we report the preliminary results of the investigation by MLST of a large number of AD hybrids with the aim to identify the circulating genotypes, their phylogenesis, and population genetics.MethodsA total of 50 AD-hybrid isolates from different parts of the world and from different sources were genotyped by MLST. Minimum spanning trees using GoeBurst algorithm were generated by comparing hybrid genotypes and by comparing separately either allele-A and allele-D portions of the hybrid genotypes to the haplotypes recorded in the MLST global database.ResultsAnalysis identified 32 hybrid genotypes grouped in three distinct main clusters (CC12, CC21, and CC30) including 12 isolates each. Both CC12 and CC21 clusters included isolates from different countries and continents but the former grouped only isolates with mating type aADalpha whereas the latter those with mating type alphaADa. Cluster CC30 included only isolates from Ivory Coasts. Heterozygous allelic combinations in each of the seven MLST loci presented two or three combinations more frequent than the other ones. In some isolates, one or more alleles were not amplified after multiple attempts, and therefore, they were considered as lacking. A total of 22 MLST profiles were identified by analyzing separately the allele-A combinations of the hybrids. Comparison with all MLST profiles of VNI, VNII, and VNB included in the MLST global database showed that the allele-A portion of the hybrid genotypes was grouped in few VNI or VNB clusters. In none of the investigated hybrids, the allele-A portion originated from VNII genotypes. Similarly, when the MLST profile of allele-D portion of hybrids was compared to all VNIV genotypes present in the global MLST database, few clusters were identified but, in this case, mostly originated from genotypes not yet found among VNIV haplotypes.ConclusionsThese preliminary results suggest that the AD hybrids here investigated originated from the mating of A haploids very common in both clinical and environmental isolates and D haploids that are not circulating at present or are very rare. Therefore, it is likely that hybrids originated in the environment where VNIV genotypic diversity is higher and suitable AD combinations can occur. Sequencing of further AD hybrids is in progress to confirm these results.

Microbiological Characterization of Cutibacterium acnes Strains Isolated from Prosthetic Joint Infections.

Aims: This study aimed to characterize 79 Cutibacterium acnes strains isolated from prosthetic joint infections (PJIs) originated from eight European hospitals. Methods: Isolates were phylotyped according to the single-locus sequence typing (SLST) scheme. We evaluated the ability of the biofilm formation of C. acnes strains isolated from PJIs and 84 isolates recovered from healthy skin. Antibiotic susceptibility testing of planktonic and biofilm cells of PJI isolates and skin isolates was performed. Results: Most of the isolates from PJIs belonged to the SLST class H/phylotype IB (34.2%), followed by class D/phylotype IA1 (21.5%), class A/phylotype IA1 (18.9%), and class K/phylotype II (13.9%). All tested isolates were biofilm producers; no difference in biofilm formation was observed between the healthy skin group and the PJI group of strains. Planktonic and sessile cells of C. acnes remained highly susceptible to a broad spectrum of antibiotics, including beta-lactams, clindamycin, fluoroquinolones, linezolid, rifampin, and vancomycin. The minimal inhibitory concentrations (MICs) for planktonic and biofilm states coincided in most cases. However, the minimal biofilm eradication concentration (MBEC) was high for all antimicrobial drugs tested (>32 mg/L), except for rifampin (2 mg/L). Conclusions: C. acnes strains isolated from healthy skin were able to produce biofilm to the same extent as isolates recovered from PJIs. All C. acnes strains in planktonic and sessile states were susceptible to most antibiotics commonly used for PJI treatment, although rifampin was the only antimicrobial agent able to eradicate C. acnes embedded in biofilm.

Analysis of bacterial diversity and genetic evolution of Lacticaseibacillus paracasei isolates in fermentation pit mud.

Since little is known about the genetic diversity of lactic acid bacteria (LAB) isolates from the fermentation pit mud (FPM), we sought to evaluate the bacterial structure, identify the LAB isolates and investigate the genotype and genetic diversity of the LAB isolates. Using high-throughput MiSeq sequencing, we identified seven dominant bacterial genera in FPM. Lactobacillus had the highest abundance. We isolated 55 LAB strains. These isolates were all identified as Lacticaseibacillus paracasei. Using an extant multilocus sequence typing (MLST) scheme, isolates were assigned to 18 sequence types (STs) and three clonal complexes. ST1, the largest group, mainly comprised FPM isolates. Niche-specific ST2 to ST18 only contained FPM isolates. Isolates could be divided into four lineages, with most assigned to Lineage 1. Only one FPM isolate was classified as L. paracasei subsp. paracasei. Other isolates could not be classified at the subspecies level using the seven MLST loci. Lactobacilli account for a high proportion of bacteria in pit mud. Based on the traditional culture method, L. paracasei was the dominant species, and these isolates exhibit a high ethanol tolerance, high intraspecific diversity and specific genetic profiles. The study described the characterization of FPM bacterial diversity, giving an insight into the genetic diversity of L. paracasei strains present in FPM.

Genomic Characterization of Salmonella Typhimurium Isolated from Guinea Pigs with Salmonellosis in Lima, Peru.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is one of the most important foodborne pathogens that infect humans globally. The gastrointestinal tracts of animals like pigs, poultry or cattle are the main reservoirs of Salmonella serotypes. Guinea pig meat is an important protein source for Andean countries, but this animal is commonly infected by S. Typhimurium, producing high mortality rates and generating economic losses. Despite its impact on human health, food security, and economy, there is no genomic information about the S. Typhimurium responsible for the guinea pig infections in Peru. Here, we sequence and characterize 11 S. Typhimurium genomes isolated from guinea pigs from four farms in Lima-Peru. We were able to identify two genetic clusters (HC100_9460 and HC100_9757) distinguishable at the H100 level of the Hierarchical Clustering of Core Genome Multi-Locus Sequence Typing (HierCC-cgMLST) scheme with an average of 608 SNPs of distance. All sequences belonged to sequence type 19 (ST19) and HC100_9460 isolates were typed in silico as monophasic variants (1,4,[5],12:i:-) lacking the fljA and fljB genes. Phylogenomic analysis showed that human isolates from Peru were located within the same genetic clusters as guinea pig isolates, suggesting that these lineages can infect both hosts. We identified a genetic antimicrobial resistance cassette carrying the ant(3)-Ia, dfrA15, qacE, and sul1 genes associated with transposons TnAs3 and IS21 within an IncI1 plasmid in one guinea pig isolate, while antimicrobial resistance genes (ARGs) for β-lactam (blaCTX-M-65) and colistin (mcr-1) resistance were detected in Peruvian human-derived isolates. The presence of a virulence plasmid highly similar to the pSLT plasmid (LT2 reference strain) containing the spvRABCD operon was found in all guinea pig isolates. Finally, seven phage sequences (STGP_Φ1 to STGP_Φ7) were identified in guinea pig isolates, distributed according to the genetic lineage (H50 clusters level) and forming part of the specific gene content of each cluster. This study presents, for the first time, the genomic characteristics of S. Typhimurium isolated from guinea pigs in South America, showing particular diversity and genetic elements (plasmids and prophages) that require special attention and also broader studies in different periods of time and locations to determine their impact on human health.

Delineating Mycobacterium abscessus population structure and transmission employing high-resolution core genome multilocus sequence typing

Mycobacterium abscessus is an emerging multidrug-resistant non-tuberculous mycobacterium that causes a wide spectrum of infections and has caused several local outbreaks worldwide. To facilitate standardized prospective molecular surveillance, we established a novel core genome multilocus sequence typing (cgMLST) scheme. Whole genome sequencing data of 1991 isolates were employed to validate the scheme, re-analyze global population structure and set genetic distance thresholds for cluster detection and taxonomic identification. We confirmed and amended the nomenclature of the main dominant circulating clones and found that these also correlate well with traditional 7-loci MLST. Dominant circulating clones could be linked to a corresponding reference genome with less than 250 alleles while 99% of pairwise comparisons between epidemiologically linked isolates were below 25 alleles and 90% below 10 alleles. These thresholds can be used to guide further epidemiological investigations. Overall, the scheme will help to unravel the apparent global spread of certain clonal complexes and as yet undiscovered transmission routes.

Toxigenic Genes, Pathogenic Potential and Antimicrobial Resistance of Bacillus cereus Group Isolated from Ice Cream and Characterized by Whole Genome Sequencing.

Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, which were characterized based on multi locus variable number tandem repeats analysis (MLVA) and whole genome sequencing (WGS). The MLVA revealed that 36 strains showed different allelic profiles. Analyses of WGS data enabled the identification of three members of the B. cereus group: B. cereus sensu stricto, B. mosaicus and B. thuringiensis. Based on the multi locus sequence typing (MLST) scheme, the strains were classified in 27 sequence types (STs), including ST26 that causes food poisoning. Toxin genes’ detection revealed the presence of the genes encoding nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), cytotoxin K (cytK) and cereulide (ces) in 100%, 44%, 42% and 8% of the strains, respectively. The identification of eleven antimicrobial resistance (AMR) genes predicted the resistance to five different antimicrobials, and the resistance to beta-lactam antibiotics was confirmed with a phenotypic antimicrobial test. Taken together, the results showed that the B. cereus strains isolated from ice cream were a potential hazard for consumer safety.

A core genome multilocus sequence typing (cgMLST) analysis of Mycoplasma bovis isolates

Mycoplasma bovis (M. bovis) is an emerging major bovine pathogen, causing economic losses worldwide in the dairy and beef industry. Whole-genome sequencing (WGS) now allows high resolution for tracing clonal populations. Based on WGS, we developed the core genome multilocus sequence typing (cgMLST) scheme and applied it onto 151 genomes of clonal and non-clonal strains of M. bovis isolated from China, Australia, Israel, Denmark, Canada, and the USA. We used the complete genome of M. bovis PG45 as the reference genome. The pairwise genome comparison of these 151 genome sequences resulted in 478 cgMLST gene targets present in > 99.0 % clonal and non-clonal isolates with 100 % overlap and > 90 % sequence similarity. A total of 478 core genes were retained as cgMLST target genes of which an average of 90.4–99 % were present in 151 M. bovis genomes, while M. agalactiae (PG2) had 17.0 % and M. mycoides subsp. capri (PG3), M. ovipneumoniae (Y98), and M. arginine resulted in 0.0 % of good targets. When tested against the clonal and non-clonal strains, we found cgMLST clusters were congruent with the MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish between clonal and epidemiologically unrelated strains of the same clonal group, which could not be achieved using traditional MLST schemes. Our results showed that ninety-two M. bovis genomes from clonal group isolates had > 10 allele differences and unambiguously differentiated from unrelated outgroup strains. Additionally, cgMLST revealed that there might be several sub-clones of the emerging ST-52 clone. The cgMLST phylogenetic analysis results showed substantial agreement with geographical and temporal information. cgMLST enables the use of next-generation sequencing technology to bovine mycoplasma epidemiology at both the local and global levels. In conclusion, the novel cgMLST scheme not only showed discrimination resolution highly as compared with MLST and SNP cgMLST in sub-typing but also indicated the capability to reveal more population structure characteristics than MLST.

Development of a novel core genome MLST scheme for tracing multidrug resistant Staphylococcus capitis

Staphylococcus capitis, which causes bloodstream infections in neonatal intensive care units, is a common cause of healthcare-associated infections. Thus, a standardized high-resolution typing method to document the transmission and dissemination of multidrug-resistant S. capitis isolates is required. We aimed to establish a core genome multilocus sequence typing (cgMLST) scheme to surveil S. capitis. The cgMLST scheme was defined based on primary and validation genome sets and tested with outbreaks of linezolid-resistant isolates and a validation set. Phylogenetic analysis was performed to investigate the population structure and compare it with the result of cgMLST analysis. The S. capitis population consists of 1 dominant, NRCS-A, and 4 less common clones. In this work, a multidrug-resistant clone (L clone) with linezolid resistance is identified. With the features of type III SCCmec and multiple copies of mutations of G2576T and C2104T in the 23S rRNA, the L clone has been spreading silently across China.